Therapeutic use of anti-cs1 antibodies

ABSTRACT

The present invention is directed to antagonists of CS1 that bind to and neutralize at least one biological activity of CS1. The invention also includes a pharmaceutical composition comprising such antibodies or antigen-binding fragments thereof. The present invention also provides for a method of preventing or treating disease states, including autoimmune disorders and cancer, in a subject in need thereof, comprising administering into said subject an effective amount of such antagonists.

This application is a continuation of U.S. patent application Ser. No.10/842,011, filed May 7, 2004 and claims the benefit of priority of U.S.Provisional Application 60/469,211, filed May 8, 2003, U.S. ProvisionalApplication 60/557,620, filed Mar. 29, 2004, U.S. ProvisionalApplication 60/557,622, filed Mar. 29, 2004, and U.S. ProvisionalApplication 60/557,621, filed Mar. 29, 2004, each of which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of antagonists and antibodies in thetreatment of diseases, including diseases related to autoimmune andcancer. The invention further relates to methods for detecting,identifying and modulating these diseases.

BACKGROUND OF THE INVENTION

Increased expression of immunoglobulin is a characteristic of manydiseases. High level secretion of immunoglobulin causes a variety ofdisorders, including hypervisocity syndrome, a debilitating disorderresulting in fatigue, headaches, shortness of breath, mental confusion,chest pain, kidney damage and failure, vision problems and Raynaud'sphenomenon (poor blood circulation, particularly fingers, toes, nose andears). Cold agglutinin disease, mixed cryoglobulinemia,hypergammaglobulinemia, Sjogren's syndrome, Lichen myxedematosus, andGaucher's disease are examples of diseases associated with increasedexpression of immunoglobulins.

Increased expression of immunoglobulin targeted to self-proteins is ahallmark of autoimmune diseases. Autoimmune disease is a failure of theimmune system to recognize auto-antigens as self. In autoimmunediseases, the immune system mistakenly attacks itself, targeting cells,tissues and organs, eventually resulting in the destruction ofphysiological systems. Autoimmunity and autoimmune diseases aremulti-factorial in origin, with genetic predisposition, host factors(e.g. weakness of immunoregulatory controls, defects in suppressor Tcells, or polyclonal stimulation of B cells resistant to controls),environmental factors and antigen-driven mechanisms being implicated inthe development of autoimmunity and production of self-antibodies toself-antigens.

Gastrointestinal disorders and Systemic Lupus Erythrematosus (SLE) aretwo examples of autoimmune diseases. Inflammatory bowel disease (IBD), asubgroup of gastrointestinal disorders, is a group of incurabledisorders that affect approximately 4 million individuals worldwide. Theetiology of recurrent inflammatory bowel disease is currently unknown.Theories include an autoimmune-mediated destruction of gastrointestinalcells, including lymphocytes. Abnormal homotypic aggregation inheritable inflammatory bowel disease models has been demonstratedpreviously, and mutations in NOD2, a gene implicated in autoimmunedisorders, has been shown to predispose patients to Crohn's disease. Ni,J. et al., Immunological abnormality in C3H/HeJ mice with heritableinflammatory bowel disease, Cell Immunol. 169:7-15 (1996); Ogura, Y. etal., A frameshift mutation in NOD2 associated with susceptibility toCrohn's Disease, Nature 411: 603-606 (2001).

IBDs most often affect the small intestine and colon, but may involveany portion of the gastrointestinal tract. There are over 1 millionpeople diagnosed with IBD in the United States alone, with over 10,000new cases diagnosed annually. Because of the drastic effect in thequality of life for IBD patients, tens of thousands of lost hours areclaimed annually, equaling up to 1 billion dollars in missed work days ayear.

IBD produces a range of gastrointestinal and extraintestinal symptoms,including diarrhea, rectal bleeding, abdominal pain, weight loss, skinand eye disorders, and delayed growth and sexual maturation in children.Two types of IBD are ulcerative colitis and Crohn's disease, which sharesimilar symptoms and physiological manifestations, but differ in themanner in which they affect the digestive tract. Ulcerative colitis ischaracterized by ulcerative inflammation of all or part of the colonicmucosa, most frequently including the rectum. Its symptoms includerectal bleeding and urgency, tenesmus, and diarrhea. Ulcerative colitisis accompanied by serious short- and long-term complications. The mostserious short-term complications are fulminant colitis, toxic megacolon,and perforation. Severe long-term complications include osteoporosis andcolorectal cancer.

Crohn's disease is a chronic transmural inflammation that may affect anypart of the gastrointestinal tract, from the mouth to the anus. Crohn'sdisease is discontinuous, with unaffected areas interspersed between oneor more involved areas. Late in the disease, the mucosa develops acobblestone appearance, which results from deep longitudinal ulcerationsinterlaced with intervening normal mucosa.

Most Crohn's disease patients present with symptoms of abdominal painand tenderness, chronic or nocturnal diarrhea, rectal bleeding, weightloss, and fever. Crohn's disease evolves over time from a primarilyinflammatory disease into one of two clinical patterns: stricturing(obstructive) or penetrating (fistulizing). In the stricturing form,transmural inflammation produces fibromuscular proliferation in theintestinal wall, followed by luminal narrowing. Symptoms of obstructionbecome common as CD progresses. In the penetrating form, sinus tractsform as inflammation tunnels through the bowel wall and breaches theserosal surface, fistulizing into adjoining tissues and even through theskin.

Ulcerative colitis and Crohn's disease are generally diagnosed usingclinical, endoscopic, and histologic criteria. However, so far thetraditional diagnostic techniques have established that no singlefinding is absolutely diagnostic for one disease or the other.Furthermore, approximately 20% of patients have a clinical picture thatfalls between Crohn's disease and ulcerative colitis. Patients that fitthis profile are said to have indeterminate colitis.

IBD symptoms can greatly impact a patient's well-being, quality of life,and capacity to function. Inflammatory periods are protracted andfrequent, and depending on the severity, life crippling. Because IBD ischronic and typically has an onset before 30 years of age, patientsgenerally require lifelong treatment. The elucidation of a role fornovel proteins and compounds in disease states for identification ofpotential targets and diagnostic markers is valuable for improving thecurrent treatment of inflammatory bowel disease patients.

SLE is characterized by the production of auto-antibodies to a varietyof ubiquitous molecules, which can have pathogenic consequencesincluding damage to numerous organs and tissues, including skin, kidney,brain, and heart. The current approved treatments for SLE involvenon-specific immunosuppression and symptom control through steroids,immunosuppressive drugs, immunomodulators, and anti-malarial drugs.However, these treatment approaches result in risks of renal toxicityand early mortality. Thus, it is desirable to develop a new approachthat specifically interferes with lymphocyte activation andauto-antibody production.

Other autoimmune diseases in which increased expression ofimmunoglobulin and/or B cells play a significant role include idiopathicthrombocytopenia, rheumatoid arthritis (RA), autoimmune hemolyticanemia, and Myasthenia gravis. Evidence for the role of B cells and/orincreased immunoglobulin comes from studies with patients treated withsteroids, immunosuppressive agents, and/or anti-CD20 antibodies (whichtarget B cells). Improvement in symptoms in these diseases correlateswith a decrease in B cells and/or serum immunoglobulin, underscoring thepivotal role that B cells play in a variety of autoimmune diseases.

Increased expression of immunoglobulin can also be seen in malignantdiseases. Like autoimmune disorders, the etiology of cancer is similarlymulti-factorial in origin. Cancer, which is the second leading cause ofdeath in the United States, has been linked to mutations in DNA thatcause unrestrained growth of cells. Genetic predisposition plays a largerole in the development of many cancers, combined with environmentalfactors, such as smoking and chemical mutagenesis.

Cancer can occur in any tissue or organ of the body. Plasma cellneoplasms, including multiple myeloma, “Solitary” myeloma of bone,extramedullary plasmacytoma, plasma cell leukemia, macroglobulinemia(including Waldenstrom's macroglobulinemia), heavy-chain disease,primary amyloidosis, monoclonal gammopathy of unknown significance(MGUS) are associated with increased expression of immunoglobulins.Chronic lymphocytic leukemia (CLL), a non-plasma cell neoplasm, is alsoassociated with high levels of immunoglobulin expression.

Myelomas, or Kahler's disease, are tumors of plasma cells derived from asingle clone, which typically originates in secondary lymphoid tissueand then migrates into and resides in bone marrow tissue. Myelomascommonly affect the bone marrow and adjacent bone structures, withprimary symptoms of bone pain and pathological fractures or lesions(osteolytic bone lesions), abnormal bleeding, anemia and increasedsusceptibility to infections. Advanced stages of the disease includerenal failure, skeletal deformities, compaction of the spinal cord, andhypercalcemia. Myeloma affects bone cells by inducing osteoclastresorption of bone, hence decimating bone structure and increasingcalcium concentration in plasma. The etiology of myelomas is currentlyunknown. Linkage to radiation damage, mutations in oncogenes, familialcauses and abnormal IL6 expression have been postulated.

Multiple myelomas are plasma cell tumors with multiple origins. Multiplemyelomas account for nearly 10% of all plasma cell malignancies, and arethe most common bone tumor cancer in adults, with an annual incidentrate of 3 to 4 cases per 100,000 people. In the United States, multiplemyelomas are the second most common hematologic malignancy afterNon-Hodgkin's Lymphoma, with approximately 50,000 cases in the UnitedStates alone, and approximately 13,500 new reported cases every year.The prognosis outlook for patients diagnosed with multiple myelomas isgrim, with only several months to a year for patients with advancedforms of the disease.

Traditional treatment regions for myeloma and multiple myelomas(henceforth referred to as “myeloma”) consist of chemotherapy, radiationtherapy, and surgery. In addition, bone marrow transplantation isrecommended for patients who are otherwise in good health. The cure ratefor patients approaches 30%, and is the only method known that can curemyelomas. However, for individuals who are older or cannot tolerate bonemarrow transplantation procedures, chemotherapy is most appropriate.

Current diagnostic procedures include X rays, bone marrow aspiration,blood and urine tests (to detect the presence of the Bence Jonesprotein), and the erythrocyte sedimentation rate assay. Potential cellsurface markers in myelomatous plasma cells have also been identified,including CD38, CD9, CD10, HLA-DR, and CD20. Ruiz-Arugelles G J and SanMiguel J F, Cell Surface Markers in Multiple Myeloma, Mayo Clin. Proc.69:684-90 (1994). Other non-B-cell lineage markers include CD2, CD4,CD13, CD14, CD15, CD23, CD24, CD25, CD33, CD39, CDw40, CD41, CD45R,CD54, CD56 and CD71, as well as unclustered antigens, R1-3, PCA-1,PCA-2, PC1, 62B1, 8A, 8F6 and MM4). Ruiz-Arugelles, supra; Leo R, etal., Multiparameter analysis of normal and malignant human plasma cells,Ann. Hematol. 64:132-9 (1992). In addition, appearance of abnormalantibodies, known as M-protein, is an indicator of multiple myeloma. Theincreased production of M-protein has been linked to hyperviscositysyndrome in multiple myelomas, causing debilitating side effects,including fatigue, headaches, shortness of breath, mental confusion,chest pain, kidney damage and failure, vision problems and Raynaud'sphenomenon (poor blood circulation, particularly fingers, toes, nose andears). Cryoglobulinemia occurs when M-protein in the blood formsparticles under cold conditions. These particles can block small bloodvessels and cause pain and numbness in the toes, fingers, and otherextremities during cold weather. Prognostic indicators, such aschromosomal deletions, elevated levels of beta-2 microglobulin, serumcreatinine levels and IgA isotyping have also been studied. Tricot G, etal., Poor prognosis in Multiple Myeloma, Blood 86:4250-2 (1995).

CS1 (SLAMF7, 19A; Genbank Accession Number NM_(—)021181.3, Ref. Bolesand Mathew (2001) Immunogenetics 52:302-307; Bouchon et al., (2001) J.Immunol. 167:5517-5521; Murphy et al., (2002) Biochem. J. 361:431-436)is a member of the CD2 subset of the immunoglobulin superfamily.Molecules of the CD2 family are involved in a broad range ofimmunomodulatory functions, such as co-activation, proliferationdifferentiation, and adhesion of lymphocytes, as well as immunoglobulinsecretion, cytokine production, and NK cell cytotoxicity. Severalmembers of the CD2 family, such as CD2, CD58, and CD150, play a role orhave been proposed to play a role in a number of autoimmune andinflammatory diseases, such as psoriasis, rheumatoid arthritis, andmultiple sclerosis.

CS1 (also known as CRACC, 19A, APEX-1, and FOAP12) was isolated andcloned by Boles, K. et al. (see Immunogenetics 52: 302-307 (2001)). Ithas been reported that CS1 plays a role in NK cell-mediated cytotoxicityand lymphocyte adhesion (Bouchon, A., et al., J. of Immuno.5517-5521(2001); Murphy, J. et al., Biochem. J. 361: 431-436 (2002)).

PCT Application PCT/US00/34963 discloses a monoclonal antibody againstAPEX-1 and the use thereof for detecting the produced recombinantextracellular domain of APEX-1. However, antibodies capable ofinhibiting immunoglobulin production by B cells and/or proliferationand/or development of myelomas have not been developed and disclosed inthe above-referenced publications. Also, evidence of over-expression ofCS-1 in autoimmune disease or cancer has not been developed or disclosedin the above-referenced publications.

SUMMARY OF THE INVENTION

The elucidation of a role for novel proteins and compounds in diseasestates for identification of potential targets and diagnostic markers isvaluable for improving the current treatment of autoimmune and cancerpatients, including patients afflicted with IBD, SLE, RA and myeloma.Accordingly, provided herein are molecular targets for treatment anddiagnosis of these diseases, particularly CS1. Additionally, providedherein are antagonists that bind to and neutralize CS1, includingneutralizing antibodies such as anti-CS1 antibodies.

The present invention is based in part on our discovery that there is nosignificant CS1 protein expression detected on platelets, red bloodcells, endothelial cells (HuVECs), kidney cells, bronchial airway cells,small airway cells, prostate cells, liver cells or breast cells. CS1expression is lymphoid specific, and is detected on cells from patients,including plasma cells from multiple myeloma and plasma cell leukemiapatients. Expression is detected only on plasma cells and not detectableat significant levels on other cell types from bone marrow samples.Accordingly, the present invention has demonstrated the feasibility ofusing anti-CS1 antibodies as therapeutic agents for the treatment ofcancer, including but not limited to plasma cell neoplasms, includingmyeloma, multiple myeloma, “solitary” myeloma of bone, extramedullaryplasmacytoma, plasma cell leukemia, macroglobulinemia (includingWaldenstrom's macroglobulinemia), heavy-chain disease, primaryamyloidosis, monoclonal gammopathy of unknown significance (MGUS). Inaddition, non-plasma cell neoplasms associated with increased expressionof immunoglobulin, including chronic lymphocytic leukemia (CLL), willalso benefit from anti-CS1 therapy.

In addition, previous studies have not revealed the expression of CS1protein on in vitro PWM (pokeweed mitogen)—activated peripheral blood Bcells, subsets of memory/effector versus naïve peripheral blood B and Tlymphocytes, or CD14⁺ monocytes/macrophages from peripheral blood.Previous studies have also not revealed the role of CS1 inimmunoglobulin production. As a result, the correlation between CS1 andautoimmune diseases has not been previously established. The presentinvention is also based in part on our discovery that the CS1 RNA andprotein expression are strongly up-regulated in activated peripheralblood B cells, the cell subset responsible for auto-antibody productionand believed to play a significant role in the development of autoimmunediseases. Furthermore, the present invention has revealed thatexpression of the CS1 RNA in SLE patient peripheral blood B lymphocytesis increased in comparison to B cells from age-matched healthy adults,as well as in patients afflicted with IBD. The present invention revealsthat CS-1 is expressed on infiltrating plasma cells in rheumatoidarthritis (RA) synovium. The present invention has also revealed thatCS1 is involved in antibody production and that antibodies to CS1decrease IgM and IgG secreted by B cells from healthy adults andpatients with lupus. Subsequently, the data of the present inventionsuggest that CS1 plays an important role in the establishment ofautoimmune diseases, especially SLE, IBD, and RA. Other diseasesassociated with an increase in immunoglobulin, B cells, and/or B cellproducts would also benefit from anti-CS1 treatment, including coldagglutinin disease, immunobullous diseases (including bullouspemphigoid, pemphigus, dermatitis herpetiformis, linear IgA disease, andepidermolysis bullosa acquista), mixed cryoglobulinemia,hypergammaglobulinemia, Sjogren's syndrome, autoimmune anemia, asthma,myasthenia gravis, multiple sclerosis, myocardial or pericardialinflammation, atopic dermatitis, psoriasis, lichen myxedematosus, andGaucher's disease.

Moreover, studies have not been conducted before to examine thefeasibility of using anti-CS1 antibodies for treating autoimmunediseases and plasma cell cancers, including myeloma and plasma cellleukemia. An ideal therapeutic antibody should bind primarily to thetarget cells. Binding to other cells and tissues can cause potentialdamage to those cells and tissues and/or deplete the therapeuticantibody so that an excess amount of the antibody is required to bedelivered to the patient in order to achieve the desired treatmentefficacy. More importantly, an antibody that binds to platelets may haveside effects, such as, platelet activation (which can lead to excessiveclotting), or platelet depletion (which can lead to failure of bloodclotting). Therefore, it is usually not feasible to use an antibody as atherapeutic agent if the antibody binds to multiple cells and tissues,especially if it binds to platelets. The present invention is based inpart on our discovery that there is no significant CS1 proteinexpression detected on platelets, red blood cells, HuVECs, kidney cells,bronchial airway cells, small airway cells, prostate cells, liver cellsand breast cells. Accordingly, the present invention has demonstratedthe feasibility of using anti-CS1 antibodies as therapeutic agents forthe treatment of autoimmune diseases, and plasma cell cancers, includingmyeloma and plasma cell leukemia.

The present invention, therefore, is directed to antagonists that bindto CS1. Exemplary embodiments of such embodiments include neutralizinganti-CS1 antibodies and antibody fragments. The antibodies neutralize atleast one biological activity of CS1, wherein said antibodies bind toCS1 and are capable of at least one of the activities selected from thegroup consisting of: (a) inhibiting immunoglobulin secretion and/orproduction by lymphocytes; and (b) inducing lysis of cells that expressCS1. The antibody or antibody-fragments of the present inventioncomprise an amino acid sequence sharing at least 85% identity with anyone of SEQ ID NOS:3-26. The present invention is also directed to anantibody or an antibody fragment, wherein said antibody or antibodyfragment binds to substantially the same epitope as an antibodycomprising an amino acid sequence of any one of SEQ ID NOS:3-26.

The present invention is also directed to an antibody or an antibodyfragment, wherein said antibody comprises a mature heavy chain variableregion comprising an amino acid sequence of SEQ ID NO:3 and a maturelight chain variable region comprising an amino acid sequence of SEQ IDNO:4.

The present invention is also directed to an antibody or an antibodyfragment, wherein said antibody comprises a mature heavy chain variableregion comprising an amino acid sequence of SEQ ID NO:5 and a maturelight chain variable region comprising an amino acid sequence of SEQ IDNO:6.

The present invention is also directed to an antibody or theantigen-binding fragment thereof, wherein said antibody comprises amature heavy chain variable region comprising an amino acid sequence ofSEQ ID NO:7 and a mature light chain variable region comprising an aminoacid sequence of SEQ ID NO:8.

The present invention is also directed to a heavy chain complementaritydetermining region (CDR) of an antibody comprising an amino acidsequence of SEQ ID NOS:8, 9, 10, 14, 15, 16, 20, 21, or 22.

The present invention is also directed to a light chain complementaritydetermining region (CDR) of an antibody comprising an amino acidsequence of SEQ ID NOS:11, 12, 13, 17, 18, 19, 23, 24, 25.

The present invention is also directed to an antibody or theantigen-binding fragment thereof, wherein said antibody comprises amature heavy chain variable region comprising an amino acid sequence ofSEQ ID NO:27 and a mature light chain variable region comprising anamino acid sequence of SEQ ID NO:28.

The present invention is also directed to a heavy chain complementaritydetermining region (CDR) of an antibody comprising an amino acidsequence of SEQ ID NOS:29 and 30. The present invention is also directedto a light chain complementarity determining region (CDR) of an antibodycomprising an amino acid sequence of SEQ ID NO:30.

The present invention is also directed to a humanized antibody or theantigen-binding fragment thereof, wherein said antibody comprises amature heavy chain variable region comprising an amino acid sequence ofSEQ ID NOS:27 or 34 and a mature light chain variable region comprisingan amino acid sequence of SEQ ID NO:28 or 39. The present invention isalso directed to a heavy chain complementarity determining region (CDR)of a humanized antibody comprising an amino acid sequence of SEQ IDNO:31.

The present invention is also directed to antibodies that bind tosubstantially the same epitope of a monoclonal antibody produced by ahybridoma cell line Luc90 having ATCC accession number PTA-5091 or bindto a non-overlapping epitope of a monoclonal antibody produced by ahybridoma cell line having ATCC accession number PTA-5091.

The present invention is also directed to antibodies that bind tosubstantially the same epitope of a monoclonal antibody produced by thehybridoma cell line Luc63 assigned ATCC accession number PTA-5950, orbind to a non-overlapping epitope of a monoclonal antibody produced bythe hybridoma cell line Luc63 assigned ATCC accession number PTA-5950.

The present invention is also directed to a pharmaceutical compositioncomprising the claimed antibody and a pharmaceutical carrier.

The present invention is also directed to a method of reducingimmunoglobulin secretion by lymphocytes, comprising contacting theleukocytes or lymphocytes with an effective amount of anti-CS1 antibody.

The present invention is also directed to a method of inducingcytotoxicity of cells expressing CS1, comprising contacting said cellswith an effective amount of an antibody against CS1.

The present invention is also directed to methods of using theantagonists of the present invention, such as anti-CS1 antibodies orantibody fragments, to prevent or treat autoimmune diseases, such as SLEand IBD, and cancer, such as myeloma, in a subject in need thereof,comprising administering said subject with an effective amount of anantagonist of CS1. Preferably, these antibodies bind to substantiallythe same epitope of a monoclonal antibody produced by a hybridoma cellline having ATCC accession number PTA-5091 or ATCC accession numberPTA-5950.

The present invention also provides methods for determining the presenceor absence of a pathological cell in a patient associated withautoimmune diseases and cancer, the method comprising detecting anucleic acid comprising a sequence at least 80% identical, preferably90% identical, more preferably 95% identical, to a sequence as describedin Table 2, hereinafter referred to as CS1, in a biological sample fromthe patient, thereby determining the presence or absence of thepathological cell. The biological sample comprises isolated nucleicacids wherein the nucleic acids may be mRNA, DNA or other nucleic acids.The biological sample is tissue from an organ which is affected by thepathology, including autoimmune diseases, including SLE, RA, and IBD,and cancer, such as myeloma and plasma cell leukemia. A further step mayincorporate the amplification of nucleic acids before the step ofdetecting the nucleic acid. The detecting is of a protein encoded by thenucleic acid, the nucleic acid comprising a CS1. The detecting step maybe carried out by using a labeled nucleic acid probe. The detecting stepmay utilize a biochip comprising at sequence at least 80% identical to aCS1 sequence, preferably 90% identical, more preferably 95% identical,or detecting a polypeptide encoded by the nucleic acid. The biologicalsample may be derived from a patient who is undergoing a therapeuticregimen to treat the pathology, or is suspected of having an autoimmunedisease or cancer pathology, including myeloma and plasma cell leukemia.

Compositions are also provided, e.g., an isolated nucleic acid moleculecomprising a CS1 sequence, including, e.g., those which are labeled; anexpression vector comprising such nucleic acid; a host cell comprisingsuch expression vector; an isolated polypeptide which is encoded by sucha nucleic acid molecule comprising a CS1 sequence; or an antibody thatspecifically binds the polypeptide. In particular embodiments, theantibody is: conjugated to an effector component, is conjugated to adetectable label (including, e.g., a fluorescent label, a radioisotope,or a cytotoxic chemical), or is a humanized antibody.

BRIEF DESCRIPTION OF THE DRAWINGS Tables

Table 1 provides the results showing specific binding activities of apanel of CS1 monoclonal antibodies.

Table 2 provides the nucleic acid and amino acid sequences for CS-1.

Table 3A and 3B shows the results of anti-CS1 treatment in the reductionof IgG production by in vitro activated B-cell lymphocytes.

Table 4 provides amino acid sequences of the heavy chain variable region(SEQ ID NO: 3) and light chain variable region (SEQ ID NO: 4) of Luc90;the amino acid sequences of the heavy chain variable region (SEQ ID NO:5) and light chain variable region (SEQ ID NO: 6) of Luc63; and theamino acid sequences of the heavy chain variable region (SEQ ID NO: 7)and light chain variable region (SEQ ID NO: 8) of Luc34. SEQ ID NOS: 9,10 and 11 depict the amino acid sequences of the Luc90 heavy chain CDR1,CDR2, and CDR3, respectively. SEQ ID NOS: 12, 13, and 14 depict theamino acid sequences of the Luc90 light chain CDR1, CDR2, and CDR3,respectively. SEQ ID NOS: 15, 16, and 17 depict the amino acid sequencesof the Luc63 heavy chain CDR1, CDR2, and CDR3, respectively. SEQ ID NOS:18, 19, and 20, depict the amino acid sequences of the Luc63 light chainCDR1, CDR2, and CDR3, respectively. SEQ ID NOS: 21, 22, and 23, depictthe amino acid sequences of the Luc34 heavy chain CDR1, CDR2, and CDR3,respectively. SEQ ID NOS: 24, 25, and 26 depict the amino acid sequencesof the Luc34 light chain CDR1, CDR2, and CDR3, respectively.

Table 5 provides amino acid sequences of the heavy chain variable region(SEQ ID NO:27) and light chain variable region (SEQ ID NO:28) of Luc 63.SEQ ID NOS:29-37 depict CDR's of the heavy chain and light chainvariable region, respectively. SEQ ID NO:33 depicts the single aminoacid mutation, from NYT to NYA (italicized), in CDR3 of the heavy chainvariable region of Luc 63.

Table 6 provides amino acid sequences of the mouse heavy chain Luc 63(SEQ ID NO:38), human heavy chain variable region cDNA (SEQ ID NO:39),human JH1 cDNA (SEQ ID NO:40) and humanized Luc 63 heavy chain variableregion (SEQ ID NO:41). Also provided are amino acid sequences of themouse light chain variable region (SEQ ID NO:42), human light chainvariable region cDNA (SEQ ID NO:43) and humanized Luc 63 light chainvariable region (SEQ ID NO:44).

Table 7 provides an alignment of the VH region amino acid sequences. Theamino acid sequences of the VH regions of MuLuc63 and HuLuc63 (SEQ IDNOS:45 and 47, respectively), and the human cDNA E55 3-14 and JH1segments (SEQ ID NO:46) are shown in single letter code. The CDRsequences are based on the definition of Kabat (Sequences of Proteins ofImmunological Interest, 5th ed., National Institutes of Health,Bethesda, Md. (1991)) are underlined in the MuLuc63 VH sequence;numbering is also according to Kabat. The CDR sequences in the human VHsegment are omitted in the figure. The single underlined amino acids inthe HuLuc63 VH sequence were predicted to contact the CDR sequences andtherefore substituted with the corresponding mouse residues. Thethreonine (T) to alanine (A) mutation made in CDR2 to eliminate thepotential N-linked glycosylation site (NYT), making the heavy chain CDR2sequence of HuLuc63 EINPDSSTNATPSLKD (SEQ ID NO:95), is indicated with adouble underline.

Table 8 provides an alignment of the VL region amino acid sequences. Theamino acid sequencesof the VL regions of MuLuc63 and HuLuc63 (SEQ IDNOS:48 and 50, respectively), and the human cDNA III-2R sequence (SEQ IDNO:49) are shown in single letter code. The CDR sequences based on thedefinition of Kabat (Sequences of Proteins of Immunological Interest,5th ed., National Institutes of Health, Bethesda, Md. (1991)) areunderlined in the MuLuc63 VL sequence; numbering is also according toKabat. The CDR sequences in the human VL segment are omitted in thefigure. The single underlined amino acids in the HuLuc63 VL sequencewere predicted to contact the CDR sequences and therefore substitutedwith the corresponding mouse residue.

Table 9 lists the oligonucleotides used for the cloning of HuLuc63 VHand VL genes.

FIGURES

FIG. 1A shows CS1 expression predominantly in plasma and memory B cells.

FIG. 1B shows CS1 expression in kidney, heart, lymph node, liver, smallintestine, brain, bone marrow, skeletal muscle, spleen and lung tissuesamples.

FIG. 2A shows CS1 expression in leukocytes and other normal adulttissues.

FIG. 2B shows increased CS1 expression in multiple activated leukocytepopulations.

FIG. 3 shows the results of competition assays of anti-CS1 monoclonalantibodies.

FIG. 4 shows the relative affinity of anti-CS1 monoclonal antibodies.

FIG. 5A shows immunohistological staining of CS1-expressing cells withthree of the anti-CS1 monoclonal antibodies (Luc 23, Luc 38, and Luc63).

FIG. 5B shows immunohistological staining of inflamed tonsil withanti-CS1 and anti-CD138 monoclonal antibodies.

FIG. 5C shows immunohistological staining of synovial joint tissue froma rheumatoid arthritis patient with anti-CS1 and anti-CD138 monoclonalantibodies.

FIG. 6 shows cell surface staining of pokeweed mitogen treatedperipheral blood mononuclear cells versus unstimulated peripheral bloodmononuclear cells with three of the anti-CS1 monoclonal antibodies (Luc63, Luc34, and Luc 38.)

FIG. 7 shows the binding activity of humanized Luc 63 (wild type NYTversus deglycosylated NYA mutation) in ELISA-based CS1-binding assays.

FIG. 8 shows a three-dimensional model of the variable region ofhumanized Luc 63.

FIG. 9 shows the up-regulation of the CS1 expression in activatedperipheral blood B and T lymphocytes assayed by real time PCR analysis.

FIG. 10 shows the increased CS1 expression in SLE patient peripheralblood B lymphocytes compared to age-matched healthy adults assayed byreal-time PCR analysis.

FIG. 11 shows a graphic representation of CS-1 expression compared tonormal adult colonic epithelial cells in microarray experiments: CS-1expression is increased in patients with ulcerative colitis (n=4) andCrohn's disease (n=5) as compared to normal adult colonic epithelialcells. Numbers over the bar symbols indicate the fold change over normaladult colonic epithelial cells for each sample.

FIG. 12 shows the upregulation of CS1 expression in inflammatory boweldisease patients with real time PCR quantification. CS-1 expression isincreased in patents with ulcerative colitis (n=2) and Crohn's disease(n=3) as compared to pooled normal adult large intestine tissue samples.Numbers over the bar symbols indicate the fold change over normal adultlarge intestine tissue for each sample.

FIG. 13 shows the prominent expression of CS1 on myeloma cells.

FIGS. 14A-14H shows the expression of CS1 in bone marrow plasma cellsfrom multiple myeloma patients.

FIG. 14I shows the expression of CS1 on peripheral blood cells from apatient with plasma cell leukemia.

FIG. 15 shows the expression of CS1 on bone marrow cell subtypes.

FIG. 16 shows the inhibition of in vitro IgM secretion of activatedPBMCs by anti-CS1 monoclonal antibodies.

FIG. 17 shows the inhibition of in vitro IgM secretion of lymphocytes byanti-CS1 monoclonal antibodies compared to anti-CD2 monoclonalantibodies.

FIG. 18 shows the inhibition of in vitro IgG secretion of lymphocytes ofhealthy adult and autoimmune disease patients by anti-CS1 monoclonalantibodies.

FIG. 19A shows the inhibition of in vivo human IgG production inSCID-HuPBMC mouse model by anti-CS1 monoclonal antibodies.

FIG. 19B shows a comparison of inhibition of in vivo human IgGproduction in SCID-HuPBMC mouse model by anti-CS1 monoclonal antibodiesLuc 90 and 63.

FIG. 19C shows a summary of the inhibition of in vivo human IgGproduction in SCID-HuPBMC mouse model by anti-CS1 monoclonal antibodies.

FIG. 20 shows the induction of antibody-dependent cellular cytotoxicityby anti-CS1 monoclonal antibodies Luc90 and Luc63.

FIG. 21A shows induction of antibody-dependent cellular cytotoxicity byanti-CS1 chimeric Luc90 monoclonal antibodies which is enhanced bychimeric antibodies with decreased levels of fucose.

FIG. 21B shows induction of antibody-dependent cellular cytotoxicity ofmultiple myeloma OPM2 cells by anti-CS1 chimeric Luc90 monoclonalantibodies.

FIG. 21C shows induction of antibody-dependent cellular cytotoxicity ofmultiple myeloma L363 cells by anti-CS1 chimeric Luc90 monoclonalantibodies.

FIG. 22 shows decreased tumor volumes in mouse xenograft multiplemyeloma models treated with anti-CS1 antibodies Luc 90 and Luc 63 versusisotype control antibodies.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides novel methods for treatment of various disorders, e.g.,autoimmune disorders and various defined cancerous conditions, includingvarious forms of myeloma. Also provided are methods for the diagnosisand prognosis evaluation of such disorders, as well as methods forscreening for compositions which modulate such conditions. The presentinvention also provides methods of monitoring the therapeutic efficacyof such treatment, including the monitoring and screening of markersselectively expressed in said disorders.

In particular, identification of markers selectively expressed inautoimmune disorders, such as SLE, RA, and IBD, and cancerousconditions, such as myeloma and plasma cell leukemia, allows for use ofthat expression in diagnostic, prognostic, or therapeutic methods. Assuch, the invention defines various compositions, e.g., nucleic acids,polypeptides, antibodies, and small molecule agonists/antagonists, whichwill be useful to selectively identify those markers. The markers may beuseful for molecular characterization of subsets of the diseases, whichsubsets may actually require very different treatments. Moreover, themarkers may also be important in diseases related to autoimmunedisorders, myeloma, and plasma cell leukemia, e.g., which affect similartissues as in such conditions, or have similar mechanisms ofinduction/maintenance. For example, tumor processes or characteristicsmay also be targeted. Diagnostic and prognostic uses are made available,e.g., to subset related but distinct diseases, to differentiate stagesof autoimmune disorders myeloma, or plasma cell leukemia or to determinetreatment strategy of such conditions. The detection methods may bebased upon nucleic acid, e.g., PCR or hybridization techniques, orprotein, e.g., ELISA, imaging, IHC, etc. The diagnosis may bequalitative or quantitative, and may detect increases or decreases inexpression levels.

Definitions

The term “CS1 protein” or “CS1 polynucleotide” or “CS1-associatedtranscript” refers to nucleic acid and polypeptide polymorphic variants,alleles, mutants, and interspecies homologues that: (1) have anucleotide sequence that has greater than about 60% nucleotide sequenceidentity, 65%, 70%, 75%, 80%, 85%, 90%, preferably about 92%, 94%, 96%,97%, 98%, or 99% or greater nucleotide sequence identity, preferablyover a region of over a region of at least about 25, 50, 100, 200, 500,1000, or more nucleotides, to a nucleotide sequence of or associatedwith the CS1 gene (Table 2), binding of the CS1 gene (Table 2) tobinding partners, e.g., polyclonal antibodies, raised against animmunogen comprising an amino acid sequence encoded by a nucleotidesequence of or associated with the CS1 gene (Table 2), andconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to a nucleic acid sequence, orthe complement thereof of CS1 (Table 2) and conservatively modifiedvariants thereof; or (4) have an amino acid sequence that has greaterthan about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%,preferably 90%, 91%, 93%, 95%, 97%, 98%, or 99% or greater aminosequence identity, preferably over a region of over a region of at leastabout 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino acidsequence encoded by a nucleotide sequence of or associated with the CS1gene (Table 2). A polynucleotide or polypeptide sequence is typicallyfrom a mammal including, but not limited to, primate, e.g., human;rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or othermammal. A “CS1 polypeptide” and a “CS1 polynucleotide,” include bothnaturally occurring or recombinant forms.

A “full length” CS1 protein or nucleic acid refers to a CS1 polypeptideor polynucleotide sequence, or a variant thereof, that contains elementsnormally contained in one or more naturally occurring, wild type CS1polynucleotide or polypeptide sequences. The “full length” may be priorto, or after, various stages of post-translational processing orsplicing, including alternative splicing.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides, e.g., of a CS1protein, polynucleotide, or transcript. Such samples include, but arenot limited to, tissue isolated from primates, e.g., humans, or rodents,e.g., mice, and rats. Biological samples may also include sections oftissues such as biopsy and autopsy samples, frozen sections taken forhistologic purposes, archival samples, blood, plasma, serum, sputum,stool, tears, mucus, hair, skin, etc. Biological samples also includeexplants and primary and/or transformed cell cultures derived frompatient tissues. A biological sample is typically obtained from aeukaryotic organism, most preferably a mammal such as a primate, e.g.,chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat,mouse; rabbit; or a bird; reptile; or fish. Livestock and domesticanimals are of interest.

“Providing a biological sample” means to obtain a biological sample foruse in methods described in this invention. Most often, this will bedone by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues ormaterials, having treatment or outcome history, will be particularlyuseful.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 93%, 95%,97%, 98%, 99%, or higher identity over a specified region, when comparedand aligned for maximum correspondence over a comparison window ordesignated region) as measured using, e.g., a BLAST or BLAST 2.0sequence comparison algorithms with default parameters described below,or by manual alignment and visual inspection. Such sequences are thensaid to be “substantially identical.” This definition also refers to, ormay be applied to, the complement of a test sequence. The definitionalso includes sequences that have deletions and/or insertions,substitutions, and naturally occurring, e.g., polymorphic or allelicvariants, and man-made variants. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is about50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof contiguous positions selected from the group consisting typically offrom about 20 to 600, usually about 50 to 200, more usually about 100 to150, in which a sequence may be compared to a reference sequence of thesame number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1981) Adv. Appl. Math. 2:482-489, by the homology alignment algorithmof Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, by the searchfor similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad.Sci. USA 85:2444-2448, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel,et al. (eds. 1995 and supplements) Current Protocols in MolecularBiology Wiley).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul, et al. (1977)Nuc. Acids Res. 25:3389-3402 and Altschul, et al. (1990) J. Mol. Biol.215:403-410. BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul, et al., supra). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, e.g., for nucleotide sequences,the parameters M (reward score for a pair of matching residues;always >0) and N (penalty score for mismatching residues; always <0).For amino acid sequences, a scoring matrix is used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915-919) alignments (B) of 50, expectation (E)of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences. See, e.g., Karlin and Altschul (1993)Proc. Nat'l. Acad. Sci. USA 90:5873-5787. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be negative large numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

An indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide encoded by the first nucleic acid isimmunologically cross reactive with the antibodies raised against thepolypeptide encoded by the second nucleic acid. Thus, a polypeptide istypically substantially identical to a second polypeptide, e.g., wherethe two peptides differ only by conservative substitutions. Anotherindication that two nucleic acid sequences are substantially identicalis that the two molecules or their complements hybridize to each otherunder stringent conditions. Yet another indication that two nucleic acidsequences are substantially identical is that the same primers can beused to amplify the sequences.

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, and the like (see, e.g., the AmericanType Culture Collection catalog or web site).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least about 85% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.“Purify” or “purification” in other embodiments means removing at leastone contaminant or component from the composition to be purified. Inthis sense, purification does not require that the purified compound behomogeneous, e.g., 100% pure.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain some basic chemical structure as anaturally occurring amino acid. Amino acid mimetic refers to a chemicalcompound that has a structure that is different from the generalchemical structure of an amino acid, but that functions similarly toanother amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variant” applies to both amino acid and nucleicacid sequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated, e.g., naturally contiguous, sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG, and GCU each encode the amino acid alanine. Thus, at eachposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. Incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallysimilar molecule. Accordingly, a silent variation of a nucleic acidwhich encodes a polypeptide is implicit in a described sequence withrespect to the expression product, but not necessarily with respect toactual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions, or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds, or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution table providing functionally similar aminoacids are well known. Such conservatively modified variants are inaddition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. Typically conservativesubstitutions include for one another: 1) Alanine (A), Glycine (G); 2)Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton (1984) Proteins: Structure and Molecular PropertiesFreeman).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts, et al. (eds. 2001)Molecular Biology of the Cell (4th ed.) Garland; and Cantor and Schimmel(1980) Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules Freeman. “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that often form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of β-sheetand α-helices. “Tertiary structure” refers to the complete threedimensional structure of a polypeptide monomer. “Quaternary structure”refers to the three dimensional structure formed, usually by thenoncovalent association of independent tertiary units. Anisotropic termsare also known as energy terms.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10,12, 15, 25, 30, 40, 50, or more nucleotides in length, up to about 100nucleotides in length. Nucleic acids and polynucleotides are a polymersof any length, including longer lengths, e.g., 200, 300, 500, 1000,2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, nucleic acid analogs are included that may have at least onedifferent linkahge, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein(1992) Oligonucleotides and Analogues: A Practical Approach Oxford Univ.Press); and peptide nucleic acid backbones and linkages. Other analognucleic acids include those with positive backbones; non-ionicbackbones, and non-ribose backbones, including those described in U.S.Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7 of Sanghvi andCook (eds. 1994) Carbohydrate Modifications in Antisense Research ACSSymposium Series 580. Nucleic acids containing one or more carbocyclicsugars are also included within one definition of nucleic acids.Modifications of the ribose-phosphate backbone may be done for a varietyof reasons, e.g., to increase the stability and half-life of suchmolecules in physiological environments or as probes on a biochip.Mixtures of naturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

A variety of references disclose such nucleic acid analogs, including,e.g., phosphoramidate (Beaucage, et al. (1993) Tetrahedron 49:1925-1963and references therein; Letsinger (1970) J. Org. Chem. 35:3800-3803;Sprinzl, et al. (1977) Eur. J. Biochem. 81:579-589; Letsinger, et al.(1986) Nucl. Acids Res. 14:3487-499; Sawai, et al. (1984) Chem. Lett.805, Letsinger, et al. (1988) J. Am. Chem. Soc. 110:4470-4471; andPauwels, et al. (1986) Chemica Scripta 26:141-149), phosphorothioate(Mag, et al. (1991) Nucleic Acids Res. 19:1437-441; and U.S. Pat. No.5,644,048), phosphorodithioate (Brill, et al. (1989) J. Am. Chem. Soc.111:2321-2322), O-methylphophoroamidite linkages (see Eckstein (1992)Oligonucleotides and Analogues: A Practical Approach, Oxford Univ.Press), and peptide nucleic acid backbones and linkages (see Egholm(1992) J. Am. Chem. Soc. 114:1895-1897; Meier, et al. (1992) Chem. Int.Ed. Engl. 31:1008-1010; Nielsen (1993) Nature 365:566-568; Carlsson, etal. (1996) Nature 380:207, all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones(Denpcy, et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097-101; non-ionicbackbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141,and 4,469,863; Kiedrowski, et al. (1991) Angew. Chem. Intl. Ed. English30:423-426; Letsinger, et al. (1988) J. Am. Chem. Soc. 110:4470-4471;Letsinger, et al. (1994) Nucleoside and Nucleotide 13:1597; Chapters 2and 3 in Sanghvi and Cook (eds. 1994) Carbohydrate Modifications inAntisense Research ACS Symposium Series 580; Mesmaeker, et al. (1994)Bioorganic and Medicinal Chem. Lett. 4:395-398; Jeffs, et al. (1994) J.Biomolecular NMR 34:17; Horn, et al. (1996) Tetrahedron Left. 37:743)and non-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7 in Sanghvi and Cook (eds.1994) Carbohydrate Modifications in Antisense Research ACS SymposiumSeries 580. Nucleic acids containing one or more carbocyclic sugars arealso included within one definition of nucleic acids (see Jenkins, etal. (1995) Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs aredescribed in Rawls (page 35, Jun. 2, 1997) C&E News.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. Peptide nucleic acids have backbones madefrom repeating N-(2-aminoiethyl)-glycine units linked by peptide bonds.The different bases (purines and pyrimidines) are linked to the backboneby methylene carbonyl linkages. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in at least two advantages. The PNA backbone exhibits improvedhybridization kinetics, resulting in stronger binding between thePNA/DNA strands, than between PNA strands and DNA strands. PNAs havelarger changes in the melting temperature (T_(m)) for mismatched versusperfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C.drop in T_(m) for an internal mismatch. With the non-ionic PNA backbone,the drop is closer to 7-9° C. Similarly, due to their non-ionic nature,hybridization of the bases attached to these backbones is relativelyinsensitive to salt concentration. In addition, PNAs are not degraded bycellular enzymes, and thus can be more stable.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The depiction of a single strand also defines thesequence of the complementary strand; thus the sequences describedherein also provide the complement of the sequence. The nucleic acid maybe DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acidmay contain combinations of deoxyribo- and ribo-nucleotides, andcombinations of bases, including uracil, adenine, thymine, cytosine,guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.“Transcript” typically refers to a naturally occurring RNA, e.g., apre-mRNA, hnRNA, or mRNA. As used herein, the term “nucleoside” includesnucleotides and nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thus,e.g., the individual units of a peptide nucleic acid, each containing abase, are referred to herein as a nucleoside.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical,physiological, chemical, or other physical means. In general, labelsfall into three classes: a) isotopic labels, which may be radioactive orheavy isotopes; b) immune labels, which may be antibodies, antigens, orepitope tags; and c) colored or fluorescent dyes. The labels may beincorporated into CS1 nucleic acids, proteins, and antibodies. Forexample, the label should be capable of producing, either directly orindirectly, a detectable signal. The detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, electron-densereagents, a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme (e.g.,as commonly used in an ELISA), biotin, digoxigenin, or haptens andproteins or other entities which can be made detectable such as alkalinephosphatase, beta-galactosidase, or horseradish peroxidase. Methods areknown for conjugating the antibody to the label. See, e.g., Hunter, etal. (1962) Nature 144:945; David, et al. (1974) Biochemistry13:1014-1021; Pain, et al. (1981) J. Immunol. Meth. 40:219-230; andNygren (1982) J. Histochem. and Cytochem. 30:407-412.

An “effector” or “effector moiety” or “effector component” is a moleculethat is bound (or linked, or conjugated), either covalently, through alinker or a chemical bond, or noncovalently, through ionic, van derWaals, electrostatic, or hydrogen bonds, to an antibody. The “effector”can be a variety of molecules including, e.g., detection moietiesincluding radioactive compounds, fluorescent compounds, enzymes orsubstrates, tags such as epitope tags, toxins; activatable moieties,chemotherapeutic agents; lipases; antibiotics; chemoattracting moieties,immune modulators (micA/B), or radioisotopes, e.g., emitting “hard”beta, radiation.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,e.g., covalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the probe may be detected by detectingthe presence of the label bound to the probe. Alternatively, methodsusing high affinity interactions may achieve the same results where oneof a pair of binding partners binds to the other, e.g., biotin,streptavidin.

As used herein a “nucleic acid probe or oligonucleotide” is a nucleicacid capable of binding to a target nucleic acid of complementarysequence through one or more types of chemical bonds, usually throughcomplementary base pairing, e.g., through hydrogen bond formation. Asused herein, a probe may include natural (e.g., A, G, C, or T) ormodified bases (7-deazaguanosine, inosine, etc.). In addition, the basesin a probe may be joined by a linkage other than a phosphodiester bond,preferably one that does not functionally interfere with hybridization.Thus, e.g., probes may be peptide nucleic acids in which the constituentbases are joined by peptide bonds rather than phosphodiester linkages.Probes may bind target sequences lacking complete complementarity withthe probe sequence depending upon the stringency of the hybridizationconditions. The probes are preferably directly labeled, e.g., withisotopes, chromophores, lumiphores, chromogens, or indirectly labeled,e.g., with biotin to which a streptavidin complex may later bind. Byassaying for the presence or absence of the probe, one can detect thepresence or absence of the select sequence or subsequence. Diagnosis orprognosis may be based at the genomic level, or at the level of RNA orprotein expression.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein, or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed, or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, e.g., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, e.g., through the expression of a recombinant nucleic acidas depicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. The proteinmay be isolated or purified away from some or most of the proteins andcompounds with which it is normally associated in its wild type host,and thus may be substantially pure. An isolated protein is unaccompaniedby at least some of the material with which it is normally associated inits natural state, preferably constituting at least about 0.5%, morepreferably at least about 5% by weight of the total protein in a givensample. A substantially pure protein comprises at least about 75% byweight of the total protein, with at least about 80% being preferred,and at least about 90% being particularly preferred. The definitionincludes the production of a CS1 protein from one organism in adifferent organism or host cell. Alternatively, the protein may be madeat a significantly higher concentration than is normally seen, throughthe use of an inducible promoter or high expression promoter, such thatthe protein is made at increased concentration levels. Alternatively,the protein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions anddeletions, as discussed below.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

A “promoter” is typically an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is active underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, e.g., wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed in operable linkage to a promoter.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule selectively to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in “Overview of principles of hybridization and thestrategy of nucleic acid assays” in Tijssen (1993) Hybridization withNucleic Probes (Laboratory Techniques in Biochemistry and MolecularBiology) (vol. 24) Elsevier. Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal is typicallyat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C. For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures may varybetween about 32°-48° C. depending on primer length. For high stringencyPCR amplification, a temperature of about 62° C. is typical, althoughhigh stringency annealing temperatures can range from about 50-65° C.,depending on the primer length and specificity. Typical cycle conditionsfor both high and low stringency amplifications include a denaturationphase of 90-95° C. for 30-120 sec, an annealing phase lasting 30-120sec, and an extension phase of about 72° C. for 1-2 min. Protocols andguidelines for low and high stringency amplification reactions areprovided, e.g., in Innis, et al. (1990) PCR Protocols: A Guide toMethods and Applications Academic Press, NY.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is typically atleast twice background. Alternative hybridization and wash conditionscan be utilized to provide conditions of similar stringency. Additionalguidelines for determining hybridization parameters are provided innumerous references, e.g., Ausubel, et al. (eds. 1991 and supplements)Current Protocols in Molecular Biology Wiley.

The phrase “changes in cell morphology” or “changes in cellularcharacteristics” refers to any change in cell morphology orproliferation characteristics in vitro or in vivo, such as cellviability, cell growth, secretion of growth or chemokine factors,changes in cell morphology, gaining or losing inflammation-specificmarkers, ability to induce or suppress inflammation when injected intosuitable animal hosts, and/or induction of a disease state in suitablehosts, e.g. autoimmune disorders and cancerous conditions. See, e.g.,pp. 231-241 in Freshney (1994) Culture of Animal Cells a Manual of BasicTechnique (2d ed.) Wiley-Liss.

“Diseased cells” refers to spontaneous or induced phenotypic changesthat do not necessarily involve the uptake of new genetic material. Forexample, although myeloma formation can arise from infection with atransforming virus and incorporation of new genomic DNA, or uptake ofexogenous DNA, it can also arise spontaneously or following exposure toan agent, thereby inducing expression or alteration of an existing gene.Tumor growth is associated with phenotypic and protein expressionchanges, such as morphological changes, aberrant cell growth, and/ornonmorphological changes. See, Freshney (2000) Culture of Animal Cells:A Manual of Basic Technique (4th ed.) Wiley-Liss. Similarly, cellsaffected by autoimmune disease processes are also associated withphenotypic and protein expression changes.

By “an effective” amount of a molecule, or an antibody, or a drug orpharmacologically active agent or pharmaceutical formulation is meant asufficient amount of the molecule, antibody, drug, agent or formulationto provide the desired effect. A “subject” or “patient” is usedinterchangeably herein, which refers to a vertebrate, preferably amammal, more preferably a human.

As used herein, the term “antibody” or “immunoglobulin” refers to aprotein consisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta, epsilon andmu constant region genes, as well as the myriad immunoglobulin variableV region genes (as indicated below, there are V genes for both H-heavy-and L-light-chains). Full-length immunoglobulin “light chains” (about 25Kd or 214 amino acids) are encoded by a variable region gene, V-kappa orV-lambda, at the NH2-terminus (about 110 amino acids) and, respectively,a kappa or lambda constant region gene at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

One form of immunoglobulin constitutes the basic structural unit of anantibody. This form is a tetramer and consists of two identical pairs ofimmunoglobulin chains, each pair having one light and one heavy chain.In each pair, the light and heavy chain variable regions are togetherresponsible for binding to an antigen, and the constant regions areresponsible for the antibody effector functions. In addition to thetetrameric antibodies, immunoglobulins may exist in a variety of otherforms including, for example, Fv, Fab, and (Fab′)₂, as well asbifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al.,Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al.,Science, 242, 423-426 (1988), which are incorporated herein byreference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y.,2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986),which are incorporated herein by reference).

Antibodies also exist, e.g., as a number of well-characterized fragmentsproduced by digestion with various peptidases. Thus, e.g., pepsindigests an antibody below the disulfide linkages in the hinge region toproduce F(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region, therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially Fab with part of the hinge region (see Paul (ed. 1999)Fundamental Immunology (4th ed.) Raven. While various antibody fragmentsare defined in terms of the digestion of an intact antibody, one ofskill will appreciate that such fragments may be synthesized de novoeither chemically or by using recombinant DNA methodology. Thus, theterm antibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, or those synthesizedde novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty,et al. (1990) Nature 348:552-554).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced, or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, and/or species, or an entirelydifferent molecule which confers new properties to the chimericantibody, e.g., an enzyme, toxin, hormone, growth factor, drug, effectorfunction, chemoattractant, immune modulator, etc.; or (b) the variableregion, or a portion thereof, is altered, replaced, or exchanged with avariable region having a different or altered antigen specificity.

The term “humanized antibody” or “humanized immunoglobulin” refers to animmunoglobulin comprising a human framework, at least one and preferablyall complementarity determining regions (CDRs) from a non-humanantibody, and in which any constant region present is substantiallyidentical to a human immunoglobulin constant region, i.e., at leastabout 85-90%, preferably at least 95% identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of one or more native humanimmunoglobulin sequences. See, e.g. Queen et al., U.S. Pat. Nos.5,5301,101; 5,585,089; 5,693,762; and 6,180,370 (These and the otherU.S. patents/patent applications are incorporated by reference in theirentirety).

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many techniques known. See, e.g., Kohler andMilstein (1975) Nature 256:495-497; Kozbor, et al. (1983) ImmunologyToday 4:72; Cole, et al. (1985) pp. 77-96 in Reisfeld and Sell (1985)Monoclonal Antibodies and Myeloma Therapy Liss; Coligan (1991) CurrentProtocols in Immunology Lippincott; Harlow and Lane (1988) Antibodies: ALaboratory Manual CSH Press; and Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press. Techniques for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms such as other mammals, may be usedto express humanized antibodies. Alternatively, phage display technologycan be used to identify antibodies and heteromeric Fab fragments thatspecifically bind to selected antigens. See, e.g., McCafferty, et al.(1990) Nature 348:552-554; Marks, et al. (1992) Biotechnology10:779-783.

The term “epitope” refers to any portion (determinant) of a protein thatis capable of eliciting an immune response and being specifically boundby an antibody. Epitope determinants usually consist of active surfacegroupings of molecules such as amino acids or GAG side chains andusually have specific three-dimensional structural characteristics, aswell as specific charge characteristics. Two antibodies are said to bindto substantially the same epitope of a protein (or the overlappingepitope of a protein) if amino acid mutations in the protein that reduceor eliminate binding of one antibody also reduce or eliminate binding ofthe other antibody, and/or if the antibodies compete for binding to theprotein, i.e., binding of one antibody to the protein reduces oreliminates binding of the other antibody. The determination of whethertwo antibodies bind substantially to the same epitope is accomplished bythe methods known in the art, such as a competition assay. In conductingan antibody competition study between a control antibody (for example,one of the anti-CS1 antibodies described herein) and any test antibody,one may first label the control antibody with a detectable label, suchas, biotin, enzymatic, radioactive label, or fluorescent label to enablethe subsequent identification. A test (unlabeled) antibody that binds tosubstantially the same epitope as the control (labeled) antibody shouldbe able to block control antibody binding and thus should reduce controlantibody binding.

In an exemplary embodiment, if an antibody binds substantially to thesame epitope of a Luc monoclonal antibody (Luc monoclonal antibodiesrefer to the produced anti-CS1 monoclonal antibodies of the presentinvention), the antibody should bind to an epitope of CS1 that overlapswith the CS1 epitope that the Luc monoclonal antibody binds to. Theoverlapping region can range from one amino acid residue to severalhundred amino acid residues. This antibody should then compete withand/or block the binding of the Luc monoclonal antibody to CS1 andthereby decrease the binding of the Luc monoclonal antibody to CS1,preferably by at least about 50% in a competition assay.

The term “derived from” means “obtained from” or “produced by” or“descending from”.

CS1 Antigens and Antibodies

SEQ ID NO:2 depicts the amino acid sequences of the full-lengthwild-type human CS1. A “functionally active” CS1 fragment or derivativeexhibits one or more functional activities associated with afull-length, wild-type CS1 protein, such as antigenic or immunogenicactivity, ability to bind natural cellular substrates, etc. Thefunctional activity of CS1 proteins, derivatives and fragments can beassayed by various methods known to one skilled in the art (CurrentProtocols in Protein Science, Coligan et al., eds., John Wiley & Sons,Inc., Somerset, N.J. (1998)). For purposes herein, functionally activefragments also include those fragments that comprise one or morestructural domains of a CS1 polypeptide, such as a binding domain.Protein domains can be identified using the PFAM program (Bateman A., etal., Nucleic Acids Res. 27: 260-2 (1999)).

CS1 polypeptide derivatives typically share a certain degree of sequenceidentity or sequence similarity with SEQ ID NO:2 or a fragment thereof.CS1 derivatives can be produced by various methods known in the art. Themanipulations that result in their production can occur at the gene orprotein level. For example, a cloned CS1 gene sequence (e.g. SEQ IDNO:1) can be cleaved at appropriate sites with restrictionendonuclease(s) (Wells et al., Philos. Trans. R. Sot. London SerA 317:415 (1986)), followed by further enzymatic modification, if desired,then isolated, and ligated in vitro, and expressed to produce thedesired derivative. Alternatively, a CS1 gene can be mutated in vitro orin vivo to create and/or destroy translation, initiation, and/ortermination sequences, or to create variations in coding regions and/orto form new restriction endonuclease sites or destroy preexisting ones,to facilitate further in vitro modification. A variety of mutagenesistechniques are known in the art such as chemical mutagenesis, in vitrosite-directed mutagenesis (Carter et al., Nucl. Acids Res. 13:4331(1986)), or use of TAB linkers (available from Pfizer, Inc.).

In one aspect, the antibodies of the present invention neutralize atleast one, or preferably all, biological activities of CS1. Thebiological activities of CS1 include: 1) binding activities of itscellular substrates, such as its ligands (for instance, theseneutralizing antibodies should be capable of competing with orcompletely blocking the binding of CS1 to at least one, and preferablyall, of its ligands); 2) signaling transduction activities; and 3)cellular responses induced by CS1.

The present invention provides for the hybridoma cell lines: Luc2, Luc3,Luc15, Luc22, Luc23, Luc29, Luc32, Luc34, Luc35, Luc37, Luc38, Luc39,Luc56, Luc60, Luc63, or Luc90. The hybridoma cell line Luc90 has beendeposited with the American Type Culture Collection (ATCC) at P. O. Box1549, Manassas, Va. 20108, as accession number PTA 5091. The deposit ofthis hybridoma cell line was received by the ATCC on Mar. 26, 2003. Thehybridoma cell line Luc63.2.22, which produces the monoclonal antibodyLuc63, has also been deposited with the ATCC at the address listedabove. The deposit of the Luc63-producing hybridoma was received by theATCC on May 6, 2004, and the hybridoma was assigned accession number PTA5950.

The present invention provides for monoclonal antibodies produced by thehybridoma cell lines: Luc2, Luc3, Luc15, Luc22, Luc23, Luc29, Luc32,Luc34, Luc35, Luc37, Luc38, Luc39, Luc56, Luc60, Luc63 (ATCC AccessionNumber PTA-5950), or Luc90 (ATCC Accession Number PTA 5091). Thesemonoclonal antibodies are named as the antibodies: Luc2, Luc3, Luc15,Luc22, Luc23, Luc29, Luc32, Luc34, Luc35, Luc37, Luc38, Luc39, Luc56,Luc60, Luc63, and Luc90, respectively, hereafter.

The present invention provides for antibodies, preferably monoclonalantibodies, that bind substantially to the same epitope as any one ofthe Luc monoclonal antibodies described herein.

The present invention provides for antibodies, preferably monoclonalantibodies, that do not bind substantially to the same epitope as one ormore of the Luc monoclonal antibodies described above.

A variety of immunological screening assays for the assessment of theantibody competition can be used to identify the antibodies that bind tosubstantially the same epitope of an antibody of the present inventionor bind to a different epitope from that of an antibody of the presentinvention.

In conducting an antibody competition study between a control antibodyand any test antibody (irrespective of species or isotype), one mayfirst label the control with a detectable label, such as, biotin or anenzymatic (or even radioactive) label to enable subsequentidentification. In this case, one would pre-mix or incubate theunlabeled antibody with cells expressing the CS1 protein. The labeledantibody is then added to the pre-incubated cells. The intensity of thebound label is measured. If the labeled antibody competes with theunlabeled antibody by binding to an overlapping epitope, the intensitywill be decreased relative to the binding by negative control unlabeledantibody (a known antibody that does not bind CS1).

The assay may be any one of a range of immunological assays based uponantibody competition, and the control antibodies would be detected bymeans of detecting their label, e.g., by using streptavidin in the caseof biotinylated antibodies or by using a chromogenic substrate inconnection with an enzymatic label (such as3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) orby simply detecting a radioactive label or a fluorescence label. Anantibody that binds to the same epitope as the control antibodies willbe able to effectively compete for binding and thus will significantlyreduce (for example, by at least 50%) the control antibody binding, asevidenced by a reduction in the bound label.

The reactivity of the (labeled) control antibodies in the absence of acompletely irrelevant antibody would be the control high value. Thecontrol low value would be obtained by incubating the unlabeled testantibodies with cells expressing CS1 and then incubate the cell/antibodymixture with labeled control antibodies of exactly the same type, whencompetition would occur and reduce binding of the labeled antibodies. Ina test assay, a significant reduction in labeled antibody reactivity inthe presence of a test antibody is indicative of a test antibody thatrecognizes substantially the same epitope.

Antibodies against CS1 of all species of origins are included in thepresent invention. Non-limiting exemplary natural antibodies includeantibodies derived from human, chicken, goats, and rodents (e.g., rats,mice, hamsters and rabbits), including transgenic rodents geneticallyengineered to produce human antibodies (see, e.g., Lonberg et al.,WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al.,WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated byreference in their entirety). Natural antibodies are the antibodiesproduced by a host animal after being immunized with an antigen, such asa polypeptide, preferably a human polypeptide. In a preferredembodiment, the antibody of the present invention is an isolated naturalantibody that binds to and/or neutralizes CS1.

The genetically altered anti-CS1 antibodies should be functionallyequivalent to the above-mentioned natural antibodies. Modifiedantibodies providing improved stability or/and therapeutic efficacy arepreferred. Examples of modified antibodies include those withconservative substitutions of amino acid residues, and one or moredeletions or additions of amino acids that do not significantlydeleteriously alter the antigen binding utility. Substitutions can rangefrom changing or modifying one or more amino acid residues to completeredesign of a region as long as the therapeutic utility is maintained.Antibodies of this invention can be modified post-translationally (e.g.,acetylation, and/or phosphorylation) or can be modified synthetically(e.g., the attachment of a labeling group). Preferred geneticallyaltered antibodies are chimeric antibodies and humanized antibodies.

The chimeric antibody is an antibody having a variable region and aconstant region derived from two different antibodies, preferablyderived from separate species. Preferably, the variable region of thechimeric antibody is derived from murine and the constant region isderived from human.

In one embodiment, the murine variable regions are derived from any oneof the monoclonal antibodies described herein. In order to produce thechimeric antibodies, the portions derived from two different species(e.g., human constant region and murine variable or binding region) canbe joined together chemically by conventional techniques or can beprepared as single contiguous proteins using genetic engineeringtechniques. The DNA molecules encoding the proteins of both the lightchain and heavy chain portions of the chimeric antibody can be expressedas contiguous proteins. The method of making chimeric antibodies isdisclosed in U.S. Pat. No. 5,677,427; U.S. Pat. No. 6,120,767; and U.S.Pat. No. 6,329,508, each of which is incorporated by reference in itsentirety.

The genetically altered antibodies used in the present invention includehumanized antibodies that bind to and neutralize CS1. In one embodiment,said humanized antibody comprising CDRs of a mouse donor immunoglobulinand heavy chain and light chain frameworks and constant regions of ahuman acceptor immunoglobulin. In one example, the humanized antibodiesare the humanized versions of any one of the antibodies describedherein. The method of making humanized antibody is disclosed in U.S.Pat. Nos.: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370each of which is incorporated herein by reference in its entirety.

Anti-CS1 fully human antibodies are also included in the presentinvention. In a preferred embodiment of the present invention, saidfully human antibodies are isolated human antibodies that neutralize theactivities of CS1 described herein.

Fully human antibodies against CS1 are produced by a variety oftechniques. One example is trioma methodology. The basic approach and anexemplary cell fusion partner, SPAZ-4, for use in this approach havebeen described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg,U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666(each of which is incorporated by reference in its entirety)

Human antibodies against CS1 can also be produced from non-humantransgenic animals having transgenes encoding at least a segment of thehuman immunoglobulin locus. The production and properties of animalshaving these properties are described in detail by, see, e.g., Lonberget al., WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al.,WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated byreference in their entirety.

Various recombinant antibody library technologies may also be utilizedto produce fully human antibodies. For example, one approach is toscreen a DNA library from human B cells according to the generalprotocol outlined by Huse et al., Science 246:1275-1281 (1989).Antibodies binding CS1 or a fragment thereof are selected. Sequencesencoding such antibodies (or binding fragments) are then cloned andamplified. The protocol described by Huse is rendered more efficient incombination with phage-display technology. See, e.g., Dower et al., WO91/17271 and McCafferty et al., WO 92/01047; U.S. Pat. No. 5,969,108,(each of which is incorporated by reference in its entirety). In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity are selected by affinity enrichment to CS1 orfragment thereof.

Eukaryotic ribosomes can also be used as means to display a library ofantibodies and isolate the binding human antibodies by screening againstthe target antigen, such as CS1, as described in Coia G, et al., J.Immunol. Methods 1: 254 (1-2):191-7 (2001); Hanes J. et al., Nat.Biotechnol. 18 (12):1287-92 (2000); Proc. Natl. Acad. Sci. U.S.A. 95(24):14130-5 (1998); Proc. Natl. Acad. Sci. U.S.A. 94 (10):4937-42(1997), each of which in incorporated by reference in its entirety.

The yeast system is also suitable for screening mammalian cell-surfaceor secreted proteins, such as antibodies. Antibody libraries may bedisplayed on the surface of yeast cells for the purpose of obtaining thehuman antibodies against a target antigen. This approach is described byYeung, et al., Biotechnol. Prog. 18(2):212-20 (2002); Boeder, E. T., etal., Nat. Biotechnol. 15(6):553-7 (1997) , each of which is hereinincorporated by reference in its entirety. Alternatively, human antibodylibraries may be expressed intracellularly and screened via yeasttwo-hybrid system (WO0200729A2, which is incorporated by reference inits entirety).

Fragments of the anti-CS1 antibodies, which retain the bindingspecificity to CS1, are also included in the present invention. Examplesof these antigen-binding fragments include, but are not limited to,partial or full heavy chains or light chains, variable regions, or CDRregions of any anti-CS1 antibodies described herein.

In a preferred embodiment of the invention, the antibody fragments aretruncated chains (truncated at the carboxyl end). Preferably, thesetruncated chains possess one or more immunoglobulin activities (e.g.,complement fixation activity). Examples of truncated chains include, butare not limited to, Fab fragments (consisting of the VL, VH, CL and CH1domains); Fd fragments (consisting of the VH and CH1 domains); Fvfragments (consisting of VL and VH domains of a single chain of anantibody); dab fragments (consisting of a VH domain); isolated CDRregions; (Fab′)₂ fragments, bivalent fragments (comprising two Fabfragments linked by a disulphide bridge at the hinge region). Thetruncated chains can be produced by conventional biochemistrytechniques, such as enzyme cleavage, or recombinant DNA techniques, eachof which is known in the art. These polypeptide fragments may beproduced by proteolytic cleavage of intact antibodies by methods wellknown in the art, or by inserting stop codons at the desired locationsin the vectors using site-directed mutagenesis, such as after CH1 toproduce Fab fragments or after the hinge region to produce (Fab′)₂fragments. Single chain antibodies may be produced by joining V_(L) andV_(H)-coding regions with a DNA that encodes a peptide linker connectingthe VL and VH protein fragments

Since the immunoglobulin-related genes contain separate functionalregions, each having one or more distinct biological activities, thegenes of the antibody fragments may be fused to functional regions fromother genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which isincorporated by reference in its entirety) to produce fusion proteins(e.g., immunotoxins) or conjugates having novel properties.

The present invention comprises the use of anti-CS1 antibodies inimmunotoxins. Conjugates that are immunotoxins including antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The conjugates of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, B. S. et al., Seminars Cell Biol 2:59-70(1991) and by Fanger, M. W. et al., Immunol Today 12:51-54 (1991).

Recombinant DNA techniques can be used to produce the recombinantanti-CS1 antibodies, as well as the chimeric or humanized anti-CS1antibodies or any other anti-CS1 genetically-altered antibodies and thefragments or conjugate thereof in any expression systems including bothprokaryotic and eukaryotic expression systems, such as bacteria, yeast,insect cells, plant cells, and mammalian cells (for example, NSO cells).

Once produced, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe purified according to stand and procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, Scopes, R., ProteinPurification (Springer-Verlag, N.Y., 1982)). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred, for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures,immunofluorescent stainings, and the like. (See, generally,Immunological Methods, Vols. I and II (Lefkovits and Pernis, eds.,Academic Press, NY, 1979 and 1981). The isolated or purified anti-CS1antibodies can be further screened for their ability of neutralizing thebiological activities of CS1 as described in the Examples.

Use of CS1 Nucleic Acids

As described above, CS1 sequences is initially identified by substantialnucleic acid and/or amino acid sequence homology or linkage to the CS1sequences of Table 2. Such homology can be based upon the overallnucleic acid or amino acid sequence, and is generally determined usingeither homology programs or hybridization conditions. Typically, linkedsequences on an mRNA are found on the same molecule.

Percent identity of a sequence can be determined using an algorithm suchas BLAST. A preferred method utilizes the BLASTN module of WU-BLAST-2set to the default parameters, with overlap span and overlap fractionset to 1 and 0.125, respectively. Alignment may include the introductionof gaps in the sequences to be aligned. In addition, for sequences whichcontain either more or fewer nucleotides than those of the nucleic acidsdescribed, the percentage of homology may be determined based on thenumber of homologous nucleosides in relation to the total number ofnucleosides. Thus, e.g., homology of sequences shorter than those of thesequences identified will be determined using the number of nucleosidesin the shorter sequence.

In one embodiment, nucleic acid homology is determined throughhybridization studies. Thus, e.g., nucleic acids which hybridize underhigh stringency to a described nucleic acid, or its complement, or isalso found on naturally occurring mRNAs is considered a homologoussequence. In another embodiment, less stringent hybridization conditionsare used; e.g., moderate or low stringency conditions may be used; seeAusubel, supra, and Tijssen, supra.

The CS1 nucleic acid sequences of the invention, e.g., the sequences inTable 2, can be fragments of larger genes, e.g., they are nucleic acidsegments. “Genes” in this context includes coding regions, non-codingregions, and mixtures of coding and non-coding regions. Accordingly,using the sequences provided herein, extended sequences, in eitherdirection, of the CS1 genes can be obtained, using techniques well knownfor cloning either longer sequences or the full length sequences; seeAusubel, et al., supra. Much can be done by informatics and manysequences can be clustered to include multiple sequences correspondingto a single gene, e.g., systems such as UniGene.

The CS1 nucleic acid of the present invention are used in several ways.In one embodiment, nucleic acid probes to CS1 are made and attached tobiochips to be used in screening and diagnostic methods, as outlinedbelow, or for administration, e.g., for gene therapy, vaccine, RNAi,and/or antisense applications. Alternatively, CS1 nucleic acids thatinclude coding regions of CS1 protein can be put into expression vectorsfor the expression of CS1 protein, again for screening purposes or foradministration to a patient.

In another embodiment, nucleic acid probes to CS1 nucleic acid (both thenucleic acid sequences outlined in the figures and/or the complementsthereof) are made. The nucleic acid probes attached to the biochip aredesigned to be substantially complementary to the CS1 nucleic acid,e.g., the target sequence (either the target sequence of the sample orto other probe sequences, e.g., in sandwich assays), such thathybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarily need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions,particularly high stringency conditions, as outlined herein.

A nucleic acid probe is generally single stranded but can be partiallysingle and partially double stranded. The strandedness of the probe isdictated by the structure, composition, and properties of the targetsequence. In general, the nucleic acid probes range from about 8-100bases long, with from about 10-80 bases being preferred, and from about30-50 bases being particularly preferred. That is, generally whole genesare not used. In some embodiments, much longer nucleic acids can beused, up to hundreds of bases.

In another embodiment, more than one probe per sequence is used, witheither overlapping probes or probes to different sections of the targetbeing used. That is, two, three, four or more probes, with three beingpreferred, are used to build in a redundancy for a particular target.The probes can be overlapping (e.g., have some sequence in common), orseparate. In some cases, PCR primers may be used to amplify signal forhigher sensitivity.

Nucleic acids can be attached or immobilized to a solid support in awide variety of ways. By “immobilized” and grammatical equivalentsherein is meant the association or binding between the nucleic acidprobe and the solid support is sufficient to be stable under theconditions of binding, washing, analysis, and removal as outlined. Thebinding can typically be covalent or non-covalent. By “non-covalentbinding” and grammatical equivalents herein is meant one or more ofelectrostatic, hydrophilic, and hydrophobic interactions. Included innon-covalent binding is the covalent attachment of a molecule, e.g.,streptavidin to the support and the non-covalent binding of thebiotinylated probe to the streptavidin. By “covalent binding” andgrammatical equivalents herein is meant that the two moieties, the solidsupport and the probe, are attached by at least one bond, includingsigma bonds, pi bonds, and coordination bonds. Covalent bonds can beformed directly between the probe and the solid support or can be formedby a cross linker or by inclusion of a specific reactive group on eitherthe solid support or the probe or both molecules. Immobilization mayalso involve a combination of covalent and non-covalent interactions.

In general, the probes are attached to the biochip in a wide variety ofways. As described herein, the nucleic acids can either be synthesizedfirst, with subsequent attachment to the biochip, or can be directlysynthesized on the biochip.

The biochip comprises a suitable solid substrate. By “substrate” or“solid support” or other grammatical equivalents herein is meant amaterial that can be modified for the attachment or association of thenucleic acid probes and is amenable to at least one detection method.Often, the substrate may contain discrete individual sites appropriatefor individual partitioning and identification. The number of possiblesubstrates is very large, and include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ,etc.), polysaccharides, nylon or nitrocellulose, resins, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses, plastics, etc. In general, the substratesallow optical detection and do not appreciably fluoresce. See WO0055627.

Generally the substrate is planar, although other configurations ofsubstrates may be used as well. For example, the probes may be placed onthe inside surface of a tube for flow-through sample analysis tominimize sample volume. Similarly, the substrate may be flexible, suchas a flexible foam, including closed cell foams made of particularplastics.

In one embodiment, the surface of the biochip and the probe may bederivatized with chemical functional groups for subsequent attachment ofthe two. Thus, e.g., the biochip is derivatized with a chemicalfunctional group including, but not limited to, amino groups, carboxygroups, oxo groups, and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the probes can beattached using functional groups on the probes. For example, nucleicacids containing amino groups can be attached to surfaces comprisingamino groups, e.g., using linkers; e.g., homo-or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200). In addition, in somecases, additional linkers, such as alkyl groups (including substitutedand heteroalkyl groups) may be used.

In this embodiment, oligonucleotides are synthesized, and then attachedto the surface of the solid support. Either the 5′ or 3′ terminus may beattached to the solid support, or attachment may be via linkage to aninternal nucleoside. In another embodiment, the immobilization to thesolid support may be very strong, yet non-covalent. For example,biotinylated oligonucleotides can be made, which bind to surfacescovalently coated with streptavidin, resulting in attachment.

Alternatively, the oligonucleotides may be synthesized on the surface.For example, photoactivation techniques utilizing photopolymerizationcompounds and techniques are used. In another embodiment, the nucleicacids can be synthesized in situ, using known photolithographictechniques, such as those described in WO 95/25116; WO 95/35505; U.S.Pat. Nos. 5,700,637 and 5,445,934; and references cited within, all ofwhich are expressly incorporated by reference; these methods ofattachment form the basis of the Affymetrix GENECHIP® (DNA Microarraychip) technology.

Often, amplification-based assays are performed to measure theexpression level of CS1-associated sequences. These assays are typicallyperformed in conjunction with reverse transcription. In such assays, aCS1-associated nucleic acid sequence acts as a template in anamplification reaction (e.g., Polymerase Chain Reaction, or PCR). In aquantitative amplification, the amount of amplification product will beproportional to the amount of template in the original sample.Comparison to appropriate controls provides a measure of the amount ofCS1-associated RNA. Methods of quantitative amplification are wellknown. Detailed protocols for quantitative PCR are provided, e.g., inInnis, et al. (1990) PCR Protocols: A Guide to Methods and ApplicationsAcademic Press.

In some embodiments, a TAQMAN® (a flurogenic oligonucleotide probe)based assay is used to measure expression. TAQMAN® based assays use afluorogenic oligonucleotide probe that contains a 5′ fluorescent dye anda 3′ quenching agent. The probe hybridizes to a PCR product, but cannotitself be extended due to a blocking agent at the 3′ end. When the PCRproduct is amplified in subsequent cycles, the 5′ nuclease activity ofthe polymerase, e.g., AMPLITAQ® (DNA polymerase), results in thecleavage of the TAQMAN® probe. This cleavage separates the 5′fluorescent dye and the 3′ quenching agent, thereby resulting in anincrease in fluorescence as a function of amplification (see, e.g.,literature provided by Perkin-Elmer).

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics4:560-569, Landegren, et al. (1988)

Science 241:1077-1080, and Barringer, et al. (1990) Gene 89:117-122),transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci.USA 86:1173-1177), self-sustained sequence replication (Guatelli, et al.(1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), dot PCR, linker adapterPCR, etc.

Expression of CS1 Protein from Nucleic Acids

In one embodiment, CS1 nucleic acid, e.g., encoding CS1 protein, areused to make a variety of expression vectors to express CS1 proteinwhich can then be used in developing reagents for diagnostic assays asdescribed below. Expression vectors and recombinant DNA technology arewell known (see, e.g., Ausubel, supra, and Fernandez and Hoeffler (eds.1999) Gene Expression Systems Academic Press) to express proteins. Theexpression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the nucleic acid encoding the CS1protein. The term “control sequences” refers to DNA sequences used forthe expression of an operably linked coding sequence in a particularhost organism. Control sequences that are suitable for prokaryotes,e.g., include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is typicallyaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, synthetic oligonucleotide adaptors or linkers are used inaccordance with conventional practice. Transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the CS1 protein. Numerous types of appropriateexpression vectors and suitable regulatory sequences are known for avariety of host cells.

In general, transcriptional and translational regulatory sequences mayinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. In one embodiment,the regulatory sequences include a promoter and transcriptional startand stop sequences.

Promoter sequences may be either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known, and are useful in the present invention.

An expression vector may comprise additional elements. For example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, e.g., in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification.Furthermore, for integrating expression vectors, the expression vectoroften contains at least one sequence homologous to the host cell genome,and preferably two homologous sequences which flank the expressionconstruct. The integrating vector may be directed to a specific locus inthe host cell by selecting the appropriate homologous sequence forinclusion in the vector. Constructs for integrating vectors areavailable. See, e.g., Fernandez and Hoeffler, supra; and Kitamura, etal. (1995) Proc. Nat'l Acad. Sci. USA 92:9146-9150.

In addition, in another embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known and will vary with the host cell used.

The CS1 protein of the present invention are usually produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding a CS1 protein, under the appropriate conditions toinduce or cause expression of the CS1 protein. Conditions appropriatefor CS1 protein expression will vary with the choice of the expressionvector and the host cell, and will be easily ascertained through routineexperimentation or optimization. For example, the use of constitutivepromoters in the expression vector will require optimizing the growthand proliferation of the host cell, while the use of an induciblepromoter requires the appropriate growth conditions for induction. Inaddition, in some embodiments, the timing of the harvest is important.For example, the baculoviral systems used in insect cell expression arelytic viruses, and thus harvest time selection can be crucial forproduct yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Saccharomyces cerevisiae and other yeasts, E. coli,Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK,CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial cells),THP1 cells (a macrophage cell line), and various other human cells andcell lines.

In one embodiment, the CS1 proteins are expressed in mammalian cells.Mammalian expression systems may be used, and include retroviral andadenoviral systems. One expression vector system is a retroviral vectorsystem such as is generally described in PCT/US97/01019 andPCT/US97/01048. Of particular use as mammalian promoters are thepromoters from mammalian viral genes, since the viral genes are oftenhighly expressed and have a broad host range. Examples include the SV40early promoter, mouse mammary tumor virus LTR promoter, adenovirus majorlate promoter, herpes simplex virus promoter, and the CMV promoter (see,e.g., Fernandez and Hoeffler, supra). Typically, transcriptiontermination and polyadenylation sequences recognized by mammalian cellsare regulatory regions located 3′ to the translation stop codon andthus, together with the promoter elements, flank the coding sequence.Examples of transcription terminator and polyadenlyation signals includethose derived from SV40.

Methods of introducing exogenous nucleic acid into mammalian hosts, aswell as other hosts, are available, and will vary with the host cellused. Techniques include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, viral infection, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In another embodiment, CS1 protein is expressed in bacterial systems.Promoters from bacteriophage may also be used. In addition, syntheticpromoters and hybrid promoters are also useful; e.g., the tac promoteris a hybrid of the trp and lac promoter sequences. Furthermore, abacterial promoter can include naturally occurring promoters ofnon-bacterial origin that have the ability to bind bacterial RNApolymerase and initiate transcription. In addition to a functioningpromoter sequence, an efficient ribosome binding site is desirable. Theexpression vector may also include a signal peptide sequence thatprovides for secretion of the CS1 protein in bacteria. The protein iseither secreted into the growth media (gram-positive bacteria) or intothe periplasmic space, located between the inner and outer membrane ofthe cell (gram-negative bacteria). The bacterial expression vector mayalso include a selectable marker gene to allow for the selection ofbacterial strains that have been transformed. Suitable selection genesinclude genes which render the bacteria resistant to drugs such asampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, andtetracycline. Selectable markers also include biosynthetic genes, suchas those in the histidine, tryptophan, and leucine biosyntheticpathways. These components are assembled into expression vectors.Expression vectors for bacteria are well known, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others (e.g., Fernandez and Hoeffler, supra). Thebacterial expression vectors are transformed into bacterial host cellsusing techniques such as calcium chloride treatment, electroporation,and others.

In one embodiment, CS1 protein is produced in insect cells using, e.g.,expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors.

In another embodiment, a CS1 protein is produced in yeast cells. Yeastexpression systems are well known, and include expression vectors forSaccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenulapolymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondiiand P. pastoris, Schizosaccharomyces pombe, and Yarrowia hpolytica.

The CS1 protein may also be made as a fusion protein, using availabletechniques. Thus, e.g., for the creation of monoclonal antibodies, ifthe desired epitope is small, the CS1 protein may be fused to a carrierprotein to form an immunogen. Alternatively, the CS1 protein may be madeas a fusion protein to increase expression, or for other reasons. Forexample, when the CS1 protein is a CS1 peptide, the nucleic acidencoding the peptide may be linked to other nucleic acid for expressionpurposes. Fusion with detection epitope tags can be made, e.g., withFLAG, His6, myc, HA, etc.

In yet another embodiment, the CS1 protein is purified or isolated afterexpression. CS1 protein may be isolated or purified in a variety of waysdepending on what other components are present in the sample and therequirements for purified product, e.g., natural conformation ordenatured. Standard purification methods include ammonium sulfateprecipitations, electrophoretic, molecular, immunological, andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the CS1 protein may be purified using a standard anti-CS1protein antibody column. Ultrafiltration and diafiltration techniques,in conjunction with protein concentration, are also useful. See, e.g.,Walsh (2002) Proteins: Biochemistry and Biotechnology Wiley; Hardin, etal. (eds. 2001) Cloning, Gene Expression and Protein Purification OxfordUniv. Press; Wilson, et al. (eds. 2000) Encyclopedia of SeparationScience Academic Press; and Scopes (1993) Protein PurificationSpringer-Verlag. The degree of purification necessary will varydepending on the use of the CS1 protein. In some instances nopurification will be necessary.

Once expressed and purified if necessary, the CS1 proteins and nucleicacids are useful in a number of applications. They may be used asimmunoselection reagents, as vaccine reagents, as screening agents,therapeutic entities, for production of antibodies, as transcription ortranslation inhibitors, etc.

Variants of CS1 Proteins

Also included within one embodiment of CS1 proteins are amino acidvariants of the naturally occurring sequences, as determined herein.Preferably, the variants are preferably greater than about 75%homologous to the wild-type sequence, more preferably greater than about80%, even more preferably greater than about 85%, and most preferablygreater than 90%. In some embodiments the homology will be as high asabout 93-95% or 98%. As for nucleic acids, homology in this contextmeans sequence similarity or identity, with identity being preferred.This homology will be determined using standard techniques, as areoutlined above for nucleic acid homologies.

CS1 protein of the present invention may be shorter or longer than thewild type amino acid sequences. Thus, in one embodiment, included withinthe definition of CS1 proteins are portions or fragments of the wildtype sequences herein. In addition, as outlined above, the CS1 nucleicacid of the invention may be used to obtain additional coding regions,and thus additional protein sequence.

In one embodiment, CS1 proteins are derivative or variant CS1 proteinsas compared to the wild-type sequence. That is, as outlined more fullybelow, the derivative CS1 peptide will often contain at least one aminoacid substitution, deletion, or insertion, with amino acid substitutionsbeing particularly preferred. The amino acid substitution, insertion, ordeletion may occur at many residue positions within the CS1 peptide.

Also included within one embodiment of CS1 proteins of the presentinvention are amino acid sequence variants. These variants typicallyfall into one or more of three classes: substitutional, insertional, ordeletional variants. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the CS1 protein,using cassette or PCR mutagenesis or other techniques, to produce DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture as outlined above. However, variant CS1 protein fragmentshaving up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques. Amino acid sequence variants arecharacterized by the predetermined nature of the variation, a featurethat sets them apart from naturally occurring allelic or interspeciesvariation of the CS1 protein amino acid sequence. The variants typicallyexhibit a similar qualitative biological activity as a naturallyoccurring analogue, although variants can also be selected which havemodified characteristics.

While the site or region for introducing an amino acid sequencevariation is often predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed CS1 variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, e.g., M13 primer mutagenesis and PCRmutagenesis. Screening of mutants is often done using assays of CS1protein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1-20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions generallyrange from about 1-20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions, or combination thereof may be usedto arrive at a final derivative. Generally these changes are done on afew amino acids to minimize the alteration of the molecule. However,larger changes may be tolerated in certain circumstances. When smallalterations in the characteristics of the CS1 protein are desired,substitutions are generally made in accordance with the amino acidsubstitution relationships described.

The variants typically exhibit essentially the same qualitativebiological activity and will elicit the same immune response as anaturally-occurring analog, although variants also are selected tomodify the characteristics of CS1 proteins as needed. Alternatively, thevariant may be designed such that a biological activity of the CS1protein is altered. For example, glycosylation sites may be added,altered, or removed.

Substantial changes in function or immunological identity are sometimesmade by selecting substitutions that are less conservative than thosedescribed above. For example, substitutions may be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example the alpha-helical or beta-sheetstructure; the charge or hydrophobicity of the molecule at the targetsite; or the bulk of the side chain. Substitutions which generally areexpected to produce the greatest changes in the polypeptide's propertiesare those in which (a) a hydrophilic residue, e.g., serine or threonineis substituted for (or by) a hydrophobic residue, e.g., leucine,isoleucine, phenylalanine, valine, or alanine; (b) a cysteine or prolineis substituted for (or by) another residue; (c) a residue having anelectropositive side chain, e.g., lysine, arginine, or histidine, issubstituted for (or by) an electronegative residue, e.g., glutamic oraspartic acid; (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine; or (e) a proline residue is incorporated or substituted,which changes the degree of rotational freedom of the peptidyl bond.

Variants typically exhibit a similar qualitative biological activity andwill elicit the same immune response as the naturally-occurring analog,although variants also are selected to modify the characteristics of theskin CS1 proteins as needed. Alternatively, the variant may be designedsuch that the biological activity of the CS1 protein is altered. Forexample, glycosylation sites may be altered or removed.

Covalent modifications of CS1 polypeptides are included within the scopeof this invention. One type of covalent modification includes reactingtargeted amino acid residues of a CS1 polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N-or C-terminal residues of a CS1 polypeptide. Derivatizationwith bifunctional agents is useful, for instance, for crosslinking CS1polypeptides to a water-insoluble support matrix or surface for use in amethod for purifying anti-CS1 polypeptide antibodies or screeningassays, as is more fully described below. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, e.g., esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-((p-azidophenyl)dithio)propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of serinyl, threonyl, or tyrosyl residues, methylationof the amino groups of the lysine, arginine, and histidine side chains(e.g., pp. 79-86, Creighton (1992) Proteins: Structure and MolecularProperties Freeman), acetylation of the N-terminal amine, and amidationof a C-terminal carboxyl group.

Another type of covalent modification of the CS1 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence CS1polypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence CS1 polypeptide. Glycosylation patternscan be altered in many ways. Different cell types to expressCS1-associated sequences can result in different glycosylation patterns.

Addition of glycosylation sites to CS1 polypeptides may also beaccomplished by altering the amino acid sequence thereof The alterationmay be made, e.g., by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence CS1 polypeptide (forO-linked glycosylation sites). The CS1 amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the CS1 polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids.

Another means of increasing the number of carbohydrate moieties on theCS1 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. See, e.g., WO 87/05330; pp. 259-306 in Aplin andWriston (1981) CRC Crit. Rev. Biochem.

Removal of carbohydrate moieties present on the CS1 polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are applicable. See,e.g., Sojar and Bahl (1987) Arch. Biochem. Biophys. 259:52-57 and Edge,et al. (1981) Anal. Biochem. 118:131-137. Enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo-and exo-glycosidases. See, e.g., Thotakura, et al.(1987) Meth. Enzymol. 138:350-359.

Another type of covalent modification of CS1 polypeptides compriseslinking the CS1 polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.

CS1 polypeptides of the present invention may also be modified in a wayto form chimeric molecules comprising a CS1 polypeptide fused to anotherheterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of a CS1 polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino-orcarboxyl-terminus of the CS1 polypeptide. The presence of suchepitope-tagged forms of a CS1 polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the CS1 polypeptide to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. In an alternative embodiment, thechimeric molecule may comprise a fusion of a CS1 polypeptide with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule, such a fusion could be to the Fcregion of an IgG molecule.

Various tag polypeptides and their respective antibodies are available.Examples include poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags; HIS6 and metal chelation tags, the flu HA tagpolypeptide and its antibody 12CA5 (Field, et al. (1988) Mol. Cell.Biol. 8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and9E10 antibodies thereto (Evan, et al. (1985) Molecular and CellularBiology 5:3610-3616); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky, et al. (1990) Protein Engineering3(6):547-553). Other tag polypeptides include the Flag-peptide (Hopp, etal. (1988) BioTechnology 6:1204-1210); the KT3 epitope peptide (Martin,et al. (1992) Science 255:192-194); tubulin epitope peptide (Skinner, etal. (1991) J. Biol. Chem. 266:15163-15166); and the T7 gene 10 proteinpeptide tag (Lutz-Freyermuth, et al. (1990) Proc. Natl. Acad. Sci. USA87:6393-6397).

Also included are CS1 proteins from other organisms, which are clonedand expressed as outlined below. Thus, probe or degenerate polymerasechain reaction (PCR) primer sequences may be used to find other relatedCS1 proteins from humans or other organisms. Particularly useful probeand/or PCR primer sequences include the unique areas of the CS1 nucleicacid sequence. Preferred PCR primers are from about 15-35 nucleotides inlength, with from about 20-30 being preferred, and may contain inosineas needed. The conditions for PCR reaction have been well described(e.g., Innis, PCR Protocols, supra).

In addition, CS1 proteins can be made that are longer than those encodedby the nucleic acids of the Table 2, e.g., by the elucidation ofextended sequences, the addition of epitope or purification tags, theaddition of other fusion sequences, etc.

CS1 proteins may also be identified as being encoded by CS1 nucleicacids. Thus, CS1 proteins are encoded by nucleic acids that willhybridize to the sequences of the sequence listings, or theircomplements, as outlined herein.

Binding Partners to CS1 Proteins CS1 Antibodies

The CS1 antibodies of the invention specifically bind to CS1 proteins.By “specifically bind” herein is meant that the antibodies bind to theprotein with a K_(d) of at least about 0.1 mM, more usually at leastabout 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better. Selectivity of binding to the specifictarget and not to related sequences is often also important.

In one embodiment, when the CS1 protein is to be used to generatebinding partners, e.g., antibodies for immunodiagnosis, the CS1 proteinshould share at least one epitope or determinant with the full lengthprotein. By “epitope” or “determinant” herein is typically meant aportion of a protein which will generate and/or bind an antibody orT-cell receptor in the context of MHC. Thus, in most instances,antibodies made to a smaller CS1 protein will be able to bind to thefull-length protein, particularly linear epitopes. In anotherembodiment, the epitope is unique; that is, antibodies generated to aunique epitope show little or no cross-reactivity. In yet anotherembodiment, the epitope is selected from a protein sequence set out inthe table.

Methods of preparing polyclonal antibodies exist (e.g., Coligan, supra;and Harlow and Lane, supra). Polyclonal antibodies can be raised in amammal, e.g., by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include a proteinencoded by a nucleic acid of Table 2 or fragment thereof or a fusionprotein thereof. It may be useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include but are not limited to keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. Examples of adjuvants which may be employed includeFreund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). Various immunization protocolsmay be used.

The antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein (1975) Nature 256:495. In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro. The immunizing agent will typically include a polypeptide encodedby a nucleic acid of the table or fragment thereof, or a fusion proteinthereof. Generally, either peripheral blood lymphocytes (“PBLs”) areused if cells of human origin are desired, or spleen cells or lymph nodecells are used if non-human mammalian sources are desired. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (e.g., pp. 59-103 in Goding (1986) Monoclonal Antibodies:Principles and Practice Academic Press). Immortalized cell lines areusually transformed mammalian cells, particularly cells of rodent,bovine, or human origin. Usually, rat or mouse cell lines are employed.The hybridoma cells may be cultured in a suitable culture medium thatpreferably contains one or more substances that inhibit the growth orsurvival of the unfused, immortalized cells. For example, if theparental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

In one embodiment, the antibodies are bispecific antibodies. Bispecificantibodies are monoclonal, preferably human or humanized, antibodiesthat have binding specificities for at least two different antigens orthat have binding specificities for two epitopes on the same antigen. Inone embodiment, one of the binding specificities is for a proteinencoded by a nucleic acid of the table or a fragment thereof, the otherone is for another antigen, and preferably for a cell-surface protein orreceptor or receptor subunit, preferably one that is CS1 specific.Alternatively, tetramer-type technology may create multivalent reagents.

In another embodiment, the antibodies have low levels or lack fucose.Antibodies lacking fucose have been correlated with enhanced ADCC(antibody-dependent cellular cytotoxicity) activity, especially at lowdoses of antibody. Shields, R. L., et al., (2002) J. Biol. Chem.277:26733-26740; Shinkawa, T. et al., (2003), J. Biol. Chem. 278:3466.Methods of preparing fucose-less antibodies include growth in ratmyeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels ofFUT8 mRNA, which encodes an enzyme (α 1,6-fucosyltransferase) necessaryfor fucosylation of polypeptides.

Alternative methods for increasing ADDC activity include mutations inthe Fc portion of a CS1 antibody, particularly mutations which increaseantibody affinity for an FcγR receptor. A correlation between increasedFcγR binding with mutated Fc has been demonstrated using targetedcytoxicity cell-based assays. Shields, R. L. et al. (2001) J. Biol. Chem276:6591-6604; Presta et al. (2002), Biochem Soc. Trans. 30:487-490.Methods for increasing ADCC activity through specific Fc regionmutations include the Fc variants comprising at least one amino acidsubstitution at a position selected from the group consisting of: 234,235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267,269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330 and 332, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat. In a preferred embodiment, said Fc variants comprise atleast one substitution selected from the group consisting of L234D,L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D,L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239D,S239E, S239N, S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T,V240M, F241W, F241L, F241Y, F241E, F241R, F243W, F243L, F243Y, F243R,F243Q, P244H, P245A, P247V, P247G, V262I, V262A, V262T, V262E, V263I,V263A, V263T, V263M, V264L, V264I, V264W, V264T, V264R, V264F, V264M,V264Y, V264E, D265G, D265N, D265Q, D265Y, D265F, D265V, D265I, D265L,D265H, D265T, V266I, V266A, V266T, V266M, S267Q, S267L, E269H, E269Y,E269F, E269R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T, Y296L, Y296I,Y296H, N297S, N297D, N297E, A298H, T299I, T299L, T299A, T299S, T299V,T299H, T299F, T299E, W313F, N325Q, N325L, N325I, N325D, N325E, N325A,N325T, N325V, N325H, A327N, A327L, L328M, L328D, L328E, L328N, L328Q,L328F, L328I, L328V, L328T, L328H, L328A, P329F, A330L, A330Y, A330V,A330I, A330F, A330R, A330H, I332D, I332E, I332N, I332Q, I332T, I332H,I332Y and I332A, wherein the numbering of the residues in the Fc regionis that of the EU index as in Kabat. Fc variants may also be selectedfrom the group consisting of V264L, V264I, F241W, F241L, F243W, F243L,F241L/F243L/V262I/V264I, F241W/F243W, F241W/F243W/V262A/V264A,F241L/V262I, F243L/V264I, F243L/V262I/V264W, F241Y/F243Y/V262T/V264T,F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,F241R/F243Q/V262T/V264R, F241E/F243Y/V262T/V264R, L328M, L328E,L328F,I332E, L3238M/I332E, P244H, P245A, P247V, W313F, P244H/P245A/P247V,P247G, V264I/I332E, F241E/F243R/V262E/V264R/I332E,F241E/F243Q/V262T/V264E/I332E, F241R/F243Q/V262T/V264R/I332E,F241E/F243Y/V262T/V264R/I332E, S298A/I332E, S239E/I332E, S239Q/I332E,S239E, D265G, D265N, S239E/D265G, S239E/D265N, S239E/D265Q, Y296E,Y296Q, T299I, A327N, S267Q/A327S, S267L/A327S, A327L, P329F, A330L,A330Y, 1332D, N297S, N297D, N297S/I332E, N297D/I332E, N297E/I332E,D265Y/N297D/I332E, D265Y/N297D/T299L/I332E, D265F/N297E/I332E,L328I/I332E,L328Q/I332E, I332N, I332Q, V264T, V264F, V240I, V263I,V266I, T299A, T299S, T299V, N325Q, N325L, N325I, S239D, S239N, S239F,S239D/I332D, S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D,S239E/I332N, S239E/I332Q, S239N/I332D, S239N/I332E, S239N/I332N,S239N/I332Q, S239Q/I332D, S239Q/I332N, S239Q/I332Q, Y296D, Y296N,F241Y/F243Y/V262T/V264T/N297D/I332E, A330Y/I332E, V264I/A330Y/I332E,A330L/I332E, V264I/A330L/I332E, L234D, L234E, L234N, L234Q, L234T,L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T,L235H, L235Y, L235I, L235V, L235F, S239T, S239H, S239Y, V240A, V240T,V240M, V263A, V263T, V263M, V264M, V264Y, V266A, V266T, V266M, E269H,E269Y, E269F, E269R, Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V,A330I, A330F, A330R, A330H, N325D, N325E, N325A, N325T, N325V, N325H,L328D/I332E, L328E/I332E, L328N/I332E, L328Q/I332E, L328V/I332E,L328T/I332E, L328H/I332E, L328I/I332E, L328A, I332T, I332H, I332Y,I332A, S239E/V264I/I332E, S239Q/V264I/I332E, S239E/V264I/A330Y/I332E,S239E/V264I/S298A/A330Y/I332E, S239D/N297D/I332E, S239E/N297D/I332E,S239D/D265V/N297D/I332E, S239D/D265I/N297D/I332E,S239D/D265L/N297D/I332E, S239D/D265F/N297D/I332E,S239D/D265Y/N297D/I332E, S239D/D265H/N297D/I332E,S239D/D265T/N297D/1332E,V264E/N297D/I332E, Y296D/N297D/I332E,Y296E/N297D/I332E, Y296N/N297D/I332E, Y296Q/N297D/I332E,Y296H/N297D/I332E, Y296T/N297D/I332E, N297D/T299V/I332E,N297D/T299I/I332E, N297D/T299L/I332E, N297D/T299F/I332E,N297D/T299H/I332E, N297D/T299E/I332E, N297D/A330Y/I332E,N297D/S298A/A330Y/I332E, S239D/A330Y/I332E,S239N/A330Y/I332E,S239D/A330L/I332E, S239N/A330L/I332E, V264I/S298A/I332E,S239D/S298A/I332E, S239N/S298A/I332E, S239D/V264I/I332E,S239D/V264I/S298A/I332E, AND S239D/264I/A330L/I332E, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat. See also PCT WO 2004/029207, Apr. 8, 2004, incorporated byreference herein.

Antibody-associated ADCC activity can be monitored and quantifiedthrough measurement of lactate dehydrogenase (LDH) release in thesupernatant, which is rapidly released upon damage to the plasmamembrane.

Other alternative embodiments for promoting cytotoxicity of cells withantibody treatment include antibody-mediated stimulation of signalingcascades resulting in cell death to the antibody bound cell. In additionantibody-mediated stimulation of the innate immune system (e.g. throughNK cells) may also result in the death of tumor cells orvirally-infected cells.

Detection of CS1 Sequence for Diagnostic Applications

In one aspect, the RNA expression levels of genes are determined fordifferent cellular states in the autoimmune disorder or cancerous, e.g.myeloma, phenotype. Expression levels of genes in normal tissue (e.g.,not undergoing a disorder) and in diseased tissue (and in some cases,for varying seventies of disorders that relate to prognosis, as outlinedbelow) are evaluated to provide expression profiles. A gene expressionprofile of a particular cell state or point of development isessentially a “fingerprint” of the state of the cell. While two statesmay have a particular gene similarly expressed, the evaluation of anumber of genes simultaneously allows the generation of a geneexpression profile that is reflective of the state of the cell. Bycomparing expression profiles of cells in different states, informationregarding which genes are important (including both up- anddown-regulation of genes) in each of these states is obtained. Then,diagnosis may be performed or confirmed to determine whether a tissuesample has the gene expression profile of normal or disesed tissue. Thiswill provide for molecular diagnosis of related conditions.

“Differential expression,” or grammatical equivalents as used herein,refers to qualitative or quantitative differences in the temporal and/orcellular gene expression patterns within and among cells and tissue.Thus, a differentially expressed gene can qualitatively have itsexpression altered, including an activation or inactivation, in, e.g.,normal versus diseased tissue. Genes may be turned on or turned off in aparticular state, relative to another state thus permitting comparisonof two or more states. A qualitatively regulated gene will exhibit anexpression pattern within a state or cell type which is detectable bystandard techniques. Some genes will be expressed in one state or celltype, but not in both. Alternatively, the difference in expression maybe quantitative, e.g., in that expression is increased or decreased;e.g., gene expression is either upregulated, resulting in an increasedamount of transcript, or downregulated, resulting in a decreased amountof transcript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques as outlinedbelow, such as by use of Affymetrix GENECHIP® (DNA microchip array)expression arrays. See, Lockhart (1996) Nature Biotechnology14:1675-1680. Other techniques include, but are not limited to,quantitative reverse transcriptase PCR, northern analysis, and RNaseprotection. As outlined above, preferably the change in expression(e.g., upregulation or downregulation) is at least about 50%, morepreferably at least about 100%, more preferably at least about 150%,more preferably at least about 200%, with from 300 to at least 1000%being especially preferred.

Evaluation may be at the gene transcript or the protein level. Theamount of gene expression may be monitored using nucleic acid probes tothe RNA or DNA equivalent of the gene transcript, and the quantificationof gene expression levels, or, alternatively, the final gene productitself (protein) can be monitored, e.g., with antibodies to CS1 proteinand standard immunoassays (ELISAs, etc.) or other techniques, includingmass spectroscopy assays, 2D gel electrophoresis assays, etc. Proteinscorresponding to CS1, e.g., those identified as being important in adisease phenotype, can be evaluated in a disease diagnostic test. Inanother embodiment, gene expression monitoring is performedsimultaneously on a number of genes. Multiple protein expressionmonitoring can be performed as well.

In this embodiment, the CS1 nucleic acid probes are attached to biochipsas outlined herein for the detection and quantification of CS1 sequencesin a particular cell. The assays are further described below in theexample. PCR techniques can be used to provide greater sensitivity.

In one embodiment nucleic acids encoding CS1 are detected. Although DNAor RNA encoding CS1 protein may be detected, of particular interest aremethods wherein an mRNA encoding a CS1 protein is detected. Probes todetect mRNA can be a nucleotide/deoxynucleotide probe that iscomplementary to and hybridizes with the mRNA and includes, but is notlimited to, oligonucleotides, cDNA, or RNA. Probes also should contain adetectable label, as defined herein. In one method the mRNA is detectedafter immobilizing the nucleic acid to be examined on a solid supportsuch as nylon membranes and hybridizing the probe with the sample.Following washing to remove the non-specifically bound probe, the labelis detected. In another method, detection of the mRNA is performed insitu. In this method permeabilized cells or tissue samples are contactedwith a detectably labeled nucleic acid probe for sufficient time toallow the probe to hybridize with the target mRNA. Following washing toremove the non-specifically bound probe, the label is detected. Forexample a digoxygenin labeled riboprobe (RNA probe) that iscomplementary to the mRNA encoding a myelomaprotein is detected bybinding the digoxygenin with an anti-digoxygenin secondary antibody anddeveloped with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate.

In another embodiment, various proteins from the three classes ofproteins as described herein (secreted, transmembrane, or intracellularproteins) are used in diagnostic assays. The CS1 proteins, antibodies,nucleic acids, modified proteins, and cells containing CS1 sequences areused in diagnostic assays. This can be performed on an individual geneor corresponding polypeptide level. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesand/or corresponding polypeptides.

As described and defined herein, CS1 protein finds use as a diseasemarker of autoimmune disorders, such as SLE, RA, and IBD, and cancerousconditions, such as myeloma and plasma cell leukemia. Additionally, CS1finds use as a marker for prognostic or diagnostic purposes. Detectionof these proteins in putative diseased tissue allows for detection,prognosis, or diagnosis of such conditions, and for selection oftherapeutic strategy. In one embodiment, antibodies are used to detectCS1. A preferred method separates proteins from a sample byelectrophoresis on a gel (typically a denaturing and reducing proteingel, but may be another type of gel, including isoelectric focusing gelsand the like). Following separation of proteins, CS1 is detected, e.g.,by immunoblotting with antibodies raised against CS1.

In another method, antibodies to CS1 find use in in situ imagingtechniques, e.g., in histology. See, e.g., Asai, et al. (eds. 1993)Methods in Cell Biology: Antibodies in Cell Biology (vol. 37) AcademicPress. In this method, cells are contacted with from one to manyantibodies to the myeloma protein(s). Following washing to removenon-specific antibody binding, the presence of the antibody orantibodies is detected. In one embodiment the antibody is detected byincubating with a secondary antibody that contains a detectable label.In another method the primary antibody to CS1 contains a detectablelabel, e.g., an enzyme marker that can act on a substrate. In anotherembodiment each one of multiple primary antibodies contains a distinctand detectable label. This method finds particular use in simultaneousscreening for CS1 along with other markers of the aforementionedconditions. Many other histological imaging techniques are also providedby the invention.

In one embodiment the label is detected in a fluorometer which has theability to detect and distinguish emissions of different wavelengths. Inaddition, a fluorescence activated cell sorter (FACS) can be used in themethod.

In another embodiment, antibodies find use in diagnosing autoimmunedisorders, such as SLE, RA, and IBD, and cancer, such as myeloma andplasma cell leukemia, from blood, serum, plasma, stool, and othersamples. Such samples, therefore, are useful as samples to be probed ortested for the presence of CS1. Antibodies can be used to detect CS1 bypreviously described immunoassay techniques including ELISA,immunoblotting (Western blotting), immunoprecipitation, BIACOREtechnology and the like. Conversely, the presence of antibodies mayindicate an immune response against an endogenous CS1 protein.

In another embodiment, in situ hybridization of labeled CS1 nucleic acidprobes to tissue arrays is done. For example, arrays of tissue samples,including diseased tissue and/or normal tissue, are made. In situhybridization (see, e.g., Ausubel, supra) is then performed. Whencomparing the fingerprints between an individual and a standard, adiagnosis, a prognosis, or a prediction may be based on the findings. Itis further understood that the genes which indicate the diagnosis maydiffer from those which indicate the prognosis and molecular profilingof the condition of the cells may lead to distinctions betweenresponsive or refractory conditions or may be predictive of outcomes.

In one embodiment, CS1 proteins, antibodies, nucleic acids, modifiedproteins, and cells containing CS1 sequences are used in prognosisassays. As above, gene expression profiles can be generated thatcorrelate to a disease state, clinical, pathological, or otherinformation, in terms of long term prognosis. Again, this may be done oneither a protein or gene level, with the use of genes being preferred.Single or multiple genes may be useful in various combinations. Asabove, CS1 probes may be attached to biochips for the detection andquantification of CS1 sequences in a tissue or patient. The assaysproceed as outlined above for diagnosis. PCR method may provide moresensitive and accurate quantification.

Genes useful in prognostic assays are genes that are differentiallyexpressed according to the stage of illness of the patient. In oneembodiment, the genes may be uniquely expressed according to the stageof the patient. In another embodiment, the genes may be expressed atdifferential levels according to the stage of the patient. For example,in myeloma, patients are accorded three different stages according tothe extent and location of the disease: Stages I, II and III. In StageI, symptoms are mild to non-existent, with many patients showing nosymptoms of myeloma. A positive diagnosis is the presence of tumorcells; however, the number of red blood cells is normal or slightlybelow normal range, there is no detectable increase in calcium in theblood, there are very low levels of M-protein in the blood or urine, andno detectable bone damage can be seen in X-rays. In Stage II, cancercells are prevalent in higher numbers. Kidney function may be affected,which worsens the prognostic diagnosis for most patients. Stage IIIbrings about anemia, hypercalcemia, advanced bone damage and high levelsof M-protein in the blood and urine. Correlation of protein expressionwith different stages in autoimmune disorder could also prove useful indetermining the prognosis of such disorders.The correlation of genesexpressed in the different stages, either uniquely expressed or havedifferential expression levels according to the stage, may be used todetermine the viability of inducing remission in a patient. This wouldbe especially useful in the earlier stages of the disease, where myelomapatients exhibit few symptoms. In addition, genes that are expressedindicating onset of long-term complications, such as beta-2microglobulin (indicator of kidney damage), as well as high levels ofserum albumin and lactate dehydrogenase, may also be useful as aprognostic tool.

The correlation of genes expressed in different stages, either uniquelyexpressed or having differential expression levels according to thestage, may also be monitored to determine the efficacy of treatmentusing the therapeutics disclosed in the present invention. For example,patients treated with antagonists of the present invention may bemonitored for therapeutic efficacy of said antagonists through themonitoring of markers, including for example, CS1 or CS1 in combinationwith disorder-specific markers (e.g. the monitoring of M-protein formyeloma treatment. Monitoring of these specific markers will beimportant in determining the efficacy of therapeutic invention, as wellas determining dosage and method of treatment considerations for thedifferent indications of the present invention.

Induction of Disease Disorders as Model Systems in Vivo InflammatoryBowel Disease

Experimental in vivo models have been developed for the investigation ofpathological processes of inflammatory bowel disease. Sartor R B,Aliment. Pharmacol. Ther. 11:89-96 (1997). For example, knock-outtransgenic mice can be made, in which the inflammatory bowel diseasegene is disrupted or in which an inflammatory bowel disease gene isinserted. Knock-out transgenic mice can be made by insertion of a markergene or other heterologous gene into the endogenous inflammatory boweldisease gene site in the mouse genome via homologous recombination. Suchmice can also be made by substituting the endogenous inflammatory boweldisease gene with a mutated version of the inflammatory bowel diseasegene, or by mutating the endogenous inflammatory bowel disease gene,e.g., by exposure to carcinogens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi, et al. (1989) Science244:1288-1292). Chimeric targeted mice can be derived according toHogan, et al. (1988) Manipulating the Mouse Embryo: A Laboratory ManualCSH Press; and Robertson (ed. 1987) Teratocarcinomas and Embryonic StemCells: A Practical Approach IRL Press, Washington, D.C.

Other models can be constructed using non-genetic manipulation of animalmodels. One model in particular has been used extensively in smallmolecule screening. This model induces colitis in rats or mice by asingle intracolonic challenge with a solution of the hapten2,4,-trinitrobenzene sulfonic acid (TNBS). Morris G P et al.,Gastroenterology 96:795-803 (1989); Boughton-Smith N K, Br. J.Pharmacol. 94:65-72 (1988). Treatment with TNBS produces an intenselocal inflammatory response that reaches its nadir after 2 to 3 days,and can last up to 21 days, depending on the severity of the challenge.

The inflammatory response produced by TNBS treatment is considered toreproduce many of the macroscopic, histological, and immunologicalhallmarks of Crohn's disease. Grisham M B et al., Gastroenterology101:540-547 (1991); Yamada Y et al., Gastroenterology 102:524-534(1992); Torres M I et al., Dig. Dis. Sci 44:2523-29 (1999); Neruath M,Fuss I, Strober W, Int. Rev. Immunol. 19:51-62 (2000). Open ulcerationis observed, with transmural inflammation and thickening of the bowelwall. Histological features include distorted crypt architecture, cryptatrophy, granulomata, giant cells, basal lymphoid aggregates, and thepresence of an inflammatory infiltrate.

The model has been used to study and validate colonic inflammation, andaddress aspects of inflammatory bowel disease. Hoffman P et al., Gut41:195-202 (1997); Jacobson K, McHugh K, Collins S M, Gastroenterology112:156-62 (1997).

Other animal models include HLA-B27 transgenic rats (Hammer RE et al.,Spontaneous inflammatory disease in transgenic rats expressing HLA-B27and Human b2M: An animal model of HLA-B27 associated human disorders,Cell 63:1088-1112 (1990)), transgenic IL-2 deficient mice (Baumgart D Cet al., Mechansisms of intestinal epithelial cell injury and colitis ininterleukin 2 deficient mice, Cell Immunol. 187:52-66 (1998)), mdrladeficient mice (Panwala C M et al., A Novel Model of Inflammatory BowelDisease: Mice deficient for the multiple drug resistance gene, mdrla,spontaneously develop colitis, J. Immunol. 161:5733-44 (1998)), and IL10 deficient mice (Freeman H J, Studies on the interleukin-10 gene inanimal models of colitis, Canadian Gastroenterology (2003)).

Myeloma

Experimental in vivo models have been developed for the investigation ofpathological processes of myeloma. Sartor R B, Aliment. Pharmacol. Ther.11:89-96 (1997). For example, knock-out transgenic mice can be made, inwhich the myeloma gene is disrupted or in which a myeloma gene isinserted. Knock-out transgenic mice can be made by insertion of a markergene or other heterologous gene into the endogenous myeloma gene site inthe mouse genome via homologous recombination. Such mice can also bemade by substituting the endogenous myeloma gene with a mutated versionof the myeloma gene, or by mutating the endogenous myeloma gene, e.g.,by exposure to carcinogens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi, et al. (1989) Science244:1288-1292). Chimeric targeted mice can be derived according toHogan, et al. (1988) Manipulating the Mouse Embryo: A Laboratory ManualCSH Press; and Robertson (ed. 1987) Teratocarcinomas and Embryonic StemCells: A Practical Approach IRL Press, Washington, D.C.

Other models can be constructed using non-genetic manipulation of animalmodels. For example, injecting C57BL/6J mice with B-cell tumors (e.g.LLC cells) can induce lung metastasis. Other animal models utilize SCIDmice and inject B-cell tumor lines (e.g. HsSultan cells (ATCC) ormultiple myeloma lines, (for example but not limited to, L363, LP-1,OPM-2, or RPMI 8226) to induce myeloma-like characteristics. Still otheranimal models include an NOD/SCID mouse model for human multiple myelomagenerated by implanting human fetal bones (FBs) into subcutaneous sitesof NOD/SCID mice, followed by inoculation with primary bone marrowmononuclear cells obtained from patients with multiple myeloma into theFBs. See Shang-Yi H., et al., Amer. J. Invest. Pathol. 164:747-756(2004). Mouse plasmacytoma models, whose formation is induced throughpristane oil (2,6,10,12-tetramethylpentadecane) treatment may also beused. In addition, mouse models in which injection of myeloma cellsdirectly into the bone marrow (orthotopic injection model) of SCID,SCID/beige or NOD/SCID mice, may also be used.

Cells undergoing transformation, as in myeloma cells, release anincreased amount of certain factors (hereinafter “myeloma specificmarkers”) than their normal counterparts. For example, CD38, CD9, CD10,HLA-DR, and CD20 are increased in expression in myeloma cells.Ruiz-Arugelles G J and San Miguel J F, Cell Surface Markers in MultipleMyeloma, Mayo Clin. Proc. 69:684-90 (1994).

Various techniques which measure the release of these factors aredescribed in Freshney (1998), supra. Also, see, Unkeless, et al. (1974)J. Biol. Chem. 249:4295-4305; Strickland and Beers (1976) J. Biol. Chem.251:5694-5702; Whur, et al. (1980) Br. J. Cancer 42:305-312; Gullino“Angiogenesis, tumor vascularization, and potential interference withtumor growth” pp. 178-184 in Mihich (ed. 1985) Biological Responses inCancer Plenum; Freshney (1985) Cancer Res. 5:111-130.

Therapeutic Methods Autoimmune Disease Treatment

In one aspect, the present invention is directed to a method of reducingthe proliferation, adhesion, differentiation, activation and/orco-activation of leukocytes, comprising contacting the leukocytes withan antagonist of CS1 described herein.

In another aspect, the present invention is directed to a method ofreducing the secretion (or production) of immunoglobulin by lymphocytes(such as B cells), comprising contacting the lymphocytes with anantagonist of CS1 described herein. Antagonists of the present inventioncan reduce the production of immunoglobulin (such as, IgM, IgG, IgD,IgA, and IgE) by at least 5%, 10%, 20%, 30%, 40%, or 50%. The percentagechange is calculated by subtracting the immunoglobulin concentrationprior to the administration of the first dose of the antibody (day 0)from the immunoglobulin concentration post dose (day x), dividing by theimmunoglobulin concentration prior to the first dose (day 0), andmultiplying by 100, e.g., [(day x−day 0)/day 0]×100.

In yet another aspect, the present invention is directed to a method ofinducing apoptosis or cytolysis of cells expressing CS1 comprisingcontacting the cells with an antibody against CS1 described herein. In apreferred embodiment, the induction is achieved via antibody-dependentcellular cytotoxicity (ADCC). In general, antibodies of the presentinvention bind antigens on the surface of target cells (cells thatexpress CS1) in the presence of effector cells (such as natural killercells or macrophages). Fc receptors on effector cells recognize boundantibodies. The cross-linking of Fc receptors signals the effector cellsto kill the target cells by cytolysis or apoptosis. Cytolysis can bedetected via detection of either the release of label or lactatedehydrogenase from the lysed cells, or detection of reduced target cellviability (e.g. annexin assay). Assays for apoptosis may be performed byterminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nickend labeling (TUNEL) assay (Lazebnik et al., Nature: 371, 346 (1994).Cytotoxicity may also be detected directly by detection kits known inthe art, such as Cytotoxicity Detection Kit from Roche Applied Science(Indianapolis, Ind.). Preferably, the antibodies of the presentinvention induce at least 10%, 20%, 30%, 40%, 50%, 60%, or 80%cytotoxicity of the target cells. The percentage is calculated by themethods disclosed in the Examples.

The antagonists can make contact with the leukocytes in vitro (such as,by adding the antagonists into a cell culture environment where theleukocytes are cultivated), ex vivo, or in vivo (for example, byadministering the antagonists into a subject).

In a preferred embodiment, the leukocytes are a) activated lymphocytes,such as B cells and/or T cells, preferably CD 19⁺ B cells and/or CD3⁺ Tcells; b) CD14⁺ activated and/or naïve cells; c) activated and/orunactivated dendritic cells; and/or c) CD56⁺ NK and/or NKT cells.

In a preferred aspect, the present invention provides for a method ofreducing the secretion of immunoglobulin by B cells, in a subject inneed thereof, comprising administering an effective amount of anantagonist of CS1 into said subject.

In another preferred aspect the present invention provides for a methodof inducing cytotoxicity, cytolysis, and/or apoptosis of cellsexpressing CS1 in a subject in need thereof, comprising administering aneffective amount of an antibody of CS1 into said subject.

The antagonists, preferably antibodies of the present invention, can beused for the prevention or treatment of autoimmune diseases, including,but not limited to, Addison's disease, autoimmune diseases of the ear,autoimmune diseases of the eye such as uveitis, autoimmune hepatitis,Crohn's disease, diabetes (Type I), epididymitis, glomerulonephritis,Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus (SLE), multiple sclerosis,myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatoid arthritis,sarcoidosis, scleroderma, psoriasis, Sjogren's syndrome,spondyloarthropathies, thyroiditis, ulcerative colitis and/orvasculitis.

In a preferred embodiment, the autoimmune disease that can be preventedand/or treated with the methods of the present invention is SLE, RA, orIBD. After being administered into a subject who has developed thesymptoms of SLE, RA, or IBD, the anti-CS1 antibodies should be able toreduce the severity of the symptoms. Alternatively, the anti-CS1antibodies can be administered to a subject before the subject developedany clinical manifestations of SLE, RA, or IBD. Such a preventiveadministration of the antibodies should completely prevent the subjectfrom developing any SLE, RA, or IBD symptoms or at least prevent thesubject from developing as severe symptoms as in the condition withoutthe antibody treatment. The severity of symptoms of SLE, RA, or IBD canbe measured by the standard clinical test for SLE, RA, or IBD known inthe art, such as serum level of anti-DNA antibodies, proteinuria, andthe mortality rate of the patients.

Therapeutic methods are usually applied to human patients but may beapplied to other mammals.

Cancer Treatment

Therapeutic methods for reducing the proliferation of myeloma cells isalso included, comprising contacting myeloma cells with an antagonist ofa myeloma protein, preferably an antibody or other antagonist, such asthe CS1 antibodies described herein. For example, the antibodies canmake contact with myeloma cells in vitro (such as, by adding theantagonists into a cell culture environment where the myeloma cells arecultivated), ex vivo, or in vivo (for example, by administering theantagonists into a subject). In another aspect, the present inventionprovides for a method of reducing the proliferation of myeloma cells,comprising administering an effective amount of a myeloma proteinantagonist into said subject.

In one aspect, the antagonists, preferably antibodies of the presentinvention, can be used for the prevention or treatment of myeloma. Afterbeing administered into a subject who has developed the symptoms ofmyeloma, the antibodies or antagonist should be able to reduce theseverity of the symptoms. Alternatively, the antibodies of the presentinvention can be administered to a subject before the subject developedany clinical manifestations of myeloma. The severity of symptoms ofmyeloma can be measured by the standard clinical test for myeloma knownin the art, such as bone-density X-ray analysis, beta-2 microglobulinlevels or hypercalcemia. Therapeutic methods are usually applied tohuman patients but may be applied to other mammals.

In yet another aspect, the present invention is directed to a method ofinducing apoptosis or cytolysis of cells expressing CS1 comprisingcontacting the cells with an antibody against CS1 described herein. In apreferred embodiment, the induction is achieved via antibody-dependentcellular cytotoxicity (ADCC). In general, antibodies of the presentinvention bind antigens on the surface of target cells (cells thatexpress CS1) in the presence of effector cells (such as natural killercells or macrophages). Fc receptors on effector cells recognize boundantibodies. The cross-linking of Fc receptors signals the effector cellsto kill the target cells by cytolysis or apoptosis. Cytolysis can bedetected via detection of either the release of label or lactatedehydrogenase from the lysed cells, or detection of reduced target cellviability (e.g. annexin assay). Assays for apoptosis may be performed byterminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nickend labeling (TUNEL) assay (Lazebnik et al., Nature: 371, 346 (1994).Cytotoxicity may also be detected directly by detection kits known inthe art, such as Cytotoxicity Detection Kit from Roche Applied Science(Indianapolis, Ind.). Preferably, the antibodies of the presentinvention induce at least 10%, 20%, 30%, 40%, 50%, 60%, or 80%cytotoxicity of the target cells. The percentage is calculated by themethods disclosed in the Examples.

The antagonists can also make contact with leukocytes in vitro (such as,by adding the antagonists into a cell culture environment where theleukocytes are cultivated), ex vivo, or in vivo (for example, byadministering the antagonists into a subject).

In a preferred embodiment, the leukocytes are a) activated lymphocytes,such as B cells and/or T cells, preferably CD19⁺ B cells and/or CD3⁺ Tcells; b) CD14⁺ activated and/or naïve cells; c) activated and/orunactivated dendritic cells; and/or c) CD56⁺ NK and/or NKT cells.

In a preferred aspect, the present invention provides for a method ofreducing the secretion of immunoglobulin by B cells, in a subject inneed thereof, comprising administering an effective amount of anantagonist of CS1 into said subject. Such reduction of immunoglobulinsecretion by B cells may help relieve complications of myeloma,including hyperviscocity syndrome.

In another preferred aspect the present invention provides for a methodof inducing cytotoxicity, cytolysis, and/or apoptosis of cellsexpressing CS1 in a subject in need thereof, comprising administering aneffective amount of an antibody of CS1 into said subject.

Administration of Therapeutic Agents

There are various methods of administering the antagonists, for example,antibodies of the present invention. Parenteral administration ispreferred. The antibody may be administered to a patient intravenouslyas a bolus or by continuous infusion over a period of time; or byintramuscular, subcutaneous, intraperitoneal, or intra-cerebrospinalroutes. Oral, topical, inhalation routes, or other delivery means knownto those skilled in the art are also included in the present invention.

The pharmaceutical compositions of the present invention commonlycomprise a solution of antagonists (for example, antibodies), or acocktail thereof dissolved in an acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers can be used, e.g., waterfor injection (WFI), or water buffered with phosphate, citrate, acetate,etc. to a pH typically of 5.0 to 8.0, most often 6.0 to 7.0, and/orcontaining salts such as sodium chloride, potassium chloride, etc. tomake isotonic. The carrier can also contain excipients such as humanserum albumin, polysorbate 80, sugars or amino acids to protect theactive protein. The concentration of an antagonist (for example,antibody) in these formulations varies widely from about 0.1 to 100mg/ml but is often in the range 1 to 10 mg/ml. The formulated monoclonalantibody is particularly suitable for parenteral administration, and canbe administered as an intravenous infusion or by subcutaneous,intramuscular or intravenous injection. Actual methods for preparingparentally administrable compositions are known or apparent to thoseskilled in the art and are described in more detail in, for example,Remington's Pharmaceutical Science (15th Ed., Mack Publishing Company,Easton, Pa., 1980), which is incorporated herein by reference. Thepresent invention provides for a pharmaceutical composition comprisingany one of the antibodies described herein.

The compositions can be administered for prophylactic and/or therapeutictreatments, comprising inhibiting the interactions between a CS1 and itscellular substrate, inhibiting the adhesion of diseased cells, orpreventing and/or reducing the clinical symptoms of the disorders above.An amount adequate to accomplish any one of these desired effects isdefined as an “effective amount”. The antibodies can be delivered into apatient by single or multiple administrations.

For the purpose of treatment of disease, the appropriate dosage of theantagonists (for example, antibodies) will depend on the severity andcourse of disease, the patient's clinical history and response, thetoxicity of the antibodies, and the discretion of the attendingphysician. The antagonists are suitably administered to the patient atone time or over a series of treatments. The initial candidate dosagemay be administered to a patient. The proper dosage and treatmentregimen can be established by monitoring the progress of therapy usingconventional techniques known to the people skilled of the art.

Additionally, the antagonist (such as antibodies) can be utilized alonein substantially pure form, or together with therapeutic agents forautoimmune diseases known to those of skill in the art. Other therapiesthat may be used in conjunction with treatment with the antibodiesinclude administration of anti-sense nucleic acid molecules orbiologicals, such as additional therapeutic antibodies. Thus, thetreatment of the present invention is formulated in a manner allowing itto be administered serially or in combination with another agent for thetreatment of autoimmune diseases. For treating autoimmune disorders andmyeloma, the antibody will often be administered after or in combinationwith one or more other immunosuppressive drugs and imunomodulators.

Kits for Use in Diagnostic and/or Prognostic Applications

For use in diagnostic and research applications suggested above, kitsare also provided by the invention. In diagnostic and researchapplications, such kits may include at least one of the following: assayreagents, buffers, CS1-specific nucleic acids or antibodies,hybridization probes and/or primers, antisense polynucleotides,ribozymes, dominant negative CS1 polypeptides or polynucleotides, smallmolecule inhibitors of CS1-associated sequences etc.

In addition, the kits may include instructional materials containinginstructions (e.g., protocols) for the practice of the methods of thisinvention. While the instructional materials typically comprise writtenor printed materials, they are not limited to such. A medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto, electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

The present invention also provides for kits for screening formodulators of CS1-associated sequences. Such kits can be prepared fromreadily available materials and reagents. For example, such kits cancomprise one or more of the following materials: a CS1-associatedpolypeptide or polynucleotide, reaction tubes, and instructions fortesting CS1-associated activity. Optionally, the kit containsbiologically active CS1 protein. A wide variety of kits and componentscan be prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user. Diagnosiswould typically involve evaluation of a plurality of genes or products.The genes will typically be selected based on correlations withimportant parameters in disease which may be identified in historical oroutcome data.

Examples Example 1 Isolation and Identification of CS1

CS1 was identified from a cDNA subtraction library of B-cell subsets(naïve vs. memory+plasma B cells) from normal healthy adult peripheralblood. CS1 was preferentially expressed among the memory and plasma Bcells. Subtraction libraries were produced by following the protocoldescribed below:

Isolation of B-Cell Subsets:

Peripheral blood mononuclear cells (PBMCs) were isolated from ninehealthy adult donors with standard Ficoll-hypaque gradients. B cellswere isolated from the PBMCs by following a standard negative selectionprotocol. PBMCs were incubated in an antibody cocktail of purified mouseanti-human CD2, CD3, CD4, CD14, CD16, CD56, CD66 and glycophorin A.After incubation and washing, goat-anti-mouse magnetic Dynal beads wereadded at 7-10 beads per cell. Subsequently, the antibody-bound cellswere isolated with a standard Dynal magnetic holder to leave enriched Bcells in the supernatants. The collected supernatants were then washedwith RPMI+10% fetal bovine serum (FBS).

Sorting of B-Cell Subsets (Naïve vs. Memory+Plasma B Cells):

Dynal-enriched B cells were stained with IgD-FITC, CD38-cychrome,CD19-APC, and CD27-PE by following the standard staining protocols. Thetwo separate populations of naïve B cells (IgD⁺CD19⁺CD38^(int/−)CD27⁻)versus memory and plasma B cells (IgD⁻ CD19⁺CD38^(int/+)CD27⁻) weresorted on a MoFlo High Performance Flow cytometer-MLS, which is equippedwith a spectra physics air-cooled argon laser (488 nm) and a 635-nmdiode laser and with filters for detection of FITC at 530/40 nm, PE at580/30 nm, APC at 670/20 nm, and cychrome (PE-Cy5) at 670/30 nm. Thesorted B cells were analyzed on the MoFlo cytometer for purity and werefound to be of 97% (memory and plasma B cells) and 98% (naïve B cells)purity. The sorted cells were placed in Trizol and stored at −70° C.

cDNA Library Production:

The cDNA subtraction libraries were made from the sorted B cell subsetsby using a standard representational difference analysis subtractivehybridization protocol. The subtraction libraries included thememory+plasma B cell cDNA library, where the naïve cDNA was subtractedtwice, and the naïve B cell cDNA library, where the memory+plasma cDNAwas subtracted twice. With standard molecular biology techniques, thecDNA subtraction library was ligated into a standard plasmid vector andtransformed into electrocompetent E. coli (DH-10B) cells. Thetransformed E. coli cells were plated on LB agar plates in the presenceof selection antibiotics. Single bacterial colonies, each representingone specific insert, were amplified using standard colony PCR.

Screening and Confirming Differerential Expression:

The cDNA subtraction library inserts were denatured and blotted onto 2identical nylon filters and hybridized separately with two differentlabeled, denatured probes−(memory+plasma)−naïve cDNA (subtracted twice)and naïve−(memory+plasma) cDNA (subtracted twice). A subtraction librarycDNA insert was considered positive if the insert selectively hybridizedpreferentially with one of the two probes. The cDNA clones for CS1hybridized preferentially with the (memory+plasma)−naïve cDNA probe(twice subtracted.)

Identification of CS1:

Bacterial cells transformed with positive clones were grown and DNA wasisolated with a Qiagen® Mini-Prep kit (in vitro diagnostic preparations)following the manufacturer's protocol (Qiagen, Valencia, Calif.). Thepurified plasmids were sequenced and the identity of the DNA sequencewas determined by searching NCBI databases.

Characterization and Confirmation of CS1 Gene Expression:

Preferential expression of the selected positive clones, including CS1,was confirmed by dot blot analysis. Equal amounts of (unsubtracted) cDNA(20 ng) isolated from sorted naïve versus memory+plasma B cells werespotted on nylon filters and hybridized with labeled cDNA insert for thepositive clone. For these assays, cDNA was synthesized from peripheralblood B cell subsets obtained from 2 separate sorts (n=9 healthy adultsand n=10 healthy adults, purity >97% and >98%, respectively). Filterswere washed and signal from hybridized probes was detected byautoradiography. A clone was considered positive if cDNA hybridizedpreferentially to the memory+plasma B cell cDNA across both sets ofsorted naïve versus memory+plasma B cells. As shown in FIG. 1A, the dataindicated that CS1 is expressed predominantly in plasma and memory Bcells.

CSI is Expressed Primarily in Lymphoid Tissue:

Dot blots were prepared from cDNA synthesized from polyA⁻RNA, which waspurchased from Clontech (Palo Alto, Calif.) and made from the followingtissues: spleen, lymph node, bone marrow, small intestine, brain, lung,skeletal muscle, heart, kidney, and liver. Dot blots were probed withdigoxygenin (DIG) labeled CS1 DNA and visualized by chemiluminescence(alkaline phosphatase labeled anti-DIG antibody and CDP-Star) followingthe manufacturer's recommendations (Boehringer-Mannheim DIG kit). Asshown in FIG. 1B, the results indicate that CS1 is expressed primarilyin lymphoid tissues (spleen, lymph node, bone marrow and smallintestine—possibly due to residual lymphocytes in Peyer's patches) andis absent or low in other non-lymphoid organs (brain, lung, skeletalmuscle, heart, kidney, and liver).

Example 2 Differential Expression of CS1 Human Cells:

Peripheral blood mononuclear cells (PBMCs) were obtained by isolationfrom standard Ficoll-hypaque gradients. Isolated PBMCs were thenresuspended at 2×10⁶ cells/ml in a fresh culture medium. PBMCs werestimulated with phytohemagglutinin (PHA) at a concentration of 3 μg/mlfor 3 days, or with pokeweed mitogen (PWM) at a concentration of 10μg/ml for 8 days. Unstimulated control PBMCs were prepared without anystimulus. Cells were cultured at 37° C. in 7% CO₂ in RPMI medium with10% FBS, penicillin, streptomycin, and glucose additives.

Mouse Cells:

Spleens were obtained from two Balb/c mice. The spleens were placed on100 micron filter. Cells were disaggregated and washed with PBS, andcentrifuged at 1,500 rpm for 10 minutes. Red blood cells were lysed with2 ml of lysis buffer at 37° C. for 2 minutes. Cells were washed twice,resuspended in 10 ml of the medium and counted. A portion of theunstimulated cells was frozen directly. The remaining cells werestimulated with con A at a concentration of 5 μg/ml for 3 days, or withLPS at a concentration of 1 μg/ml for 3 days. Cells were cultured in aDMEM medium with 10% FBS, antibiotics, and glucose additives.

B Lymphocytes from Lupus Patient versus Age Matched Healthy Individuals:

B cells were sorted from peripheral blood mononuclear cells of lupuspatient and healthy individuals, by staining the cells with FITC-labeledanti- human CD19 antibody. Cells were sorted on a MoFLo High PerformanceFlow Cytometer-MLS as described in Example 1. Cells were collected intosterile medium for RNA synthesis.

Total RNA Isolation for Real-Time PCR:

Cells were washed once and placed in Trizol™ (Life Technologies,Gaithersburg, Md.), and total RNA was isolated following themanufacturer's protocol. Total RNA was treated with RNase-free DNase(GenHunter, Nashville, Tenn.). DNase-digested RNA was extracted withphenol/chloroform and precipitated overnight with ethanol. RNA waswashed with 75% ethanol and dissolved in nuclease-free water. Theisolated RNA was quantitated and its integrity was analyzed on anagarose gel.

Real-Time PCR:

Total RNA (2 μg) was reverse-transcribed from the lupus patient's versushealthy individuals' sorted B lymphocytes in 100 μl of reaction mixtureby using standard Taqman reverse transcription reagents (AppliedBiosystems, Foster City, Calif.). PCR reactions were set up using SYBRgreen PCR master mix from Applied Biosystems. CS1 primers wereincorporated in the mix to examine the expression levels of CS1 in lupuspatient's and healthy individuals' cDNA. CS1 primers were designed fromthe published sequences (Genbank accession number XM-001635, AF390894).β-Actin and 18S rRNA primers were used as controls for normalization.The primers were designed using Primer Express software purchased fromApplied Biosystems. The PCR amplified products were 85bp for CS1primers, 84bp for β-actin primers, and 61bp for 18S rRNA primers.Real-Time PCR was performed in a GeneAmp 5700 SDS system from AppliedBiosystems, using the recommended protocol.

Real-Time PCR of Mouse Novel Ly9:

Total RNA (2 μg) was reverse transcribed from conA, LPS, andunstimulated samples in 100 μl of reaction mixture using standard Taqmanreverse transcription reagents (Applied Biosystems). A PCR reaction wasset up using SYBR green PCR master mix from Applied Biosystems. Primersspecific for mouse novel Ly9 were designed from the published sequence(Genbank accession number AF467909) and incorporated in the mix toexamine the expression levels in stimulated vs. unstimulated cDNAsamples. The 18S rRNA primers were used for normalization. The primerswere designed by using Primer Express software purchased from AppliedBiosystems. The PCR amplified products were 65bp for the mouse Ly9primers and 61bp for the 18S rRNA primers. Real-Time PCR was performedin a GeneAmp 5700 Sequence Detection System from Applied Biosystems,using the recommended protocol.

Microarray Assays: Sample Preparation, Labeling Microchips andFingerprints

Expression profiles of activated and non-activated leukocytes populatonswere determined and analyzed using gene chips. The custom AffymetrixGeneChip® oligonucleotide microarray allows interrogation ofapproximately 35,000 unique mRNA transcripts.

RNA was isolated and gene chip analysis was performed as described (SeeHenshall et al. (2003) Cancer Research 63:4196-4203; Henshall et al.(2003) Oncogene 22:6005-12; Glynne, et al. (2000) Nature 403:672-676;Zhao, et al. (2000) Genes Dev. 14:981-993, herein incorporated in itsentirety).

Purify Poly A+ mRNA from Total RNA or Clean Up Total RNA with Qiagen'sRNEASY® (Purification of Poly A⁺ mRNA from Total RNA) Kit

The oligotex suspension was heated to 37° C. and mixed immediatelybefore adding to RNA. The Elution Buffer was incubated at 70° C. Notethat the 2× Binding Buffer may be warmed up at 65° C. if there isprecipitate in the buffer. Total RNA was mixed with DEPC-treated water,2× Binding Buffer, and Oligotex according to Table 2 on page 16 of theOligotex Handbook. The mixture was incubated for 3 minutes at 65° C.,and then incubated for 10 minutes at room temperature.

The tubes were centrifuged for 2 minutes at 14,000 to 18,000 g. Notethat if the centrifuge has a “soft setting,” it should be used. Thesupernatant was removed without disturbing the Oligotex pellet. A smallamount of solution may be left behind to reduce the loss of Oligotex.The supernatant should be saved until certain that satisfactory bindingand elution of poly A⁺ mRNA has occurred.

The pellet was gently resuspended in Wash Buffer OW2 and pipetted ontothe spin column. The spin column was centrifuged at full speed (softsetting if possible) for 1 minute.

After centrifugation, the spin column was transferred to a newcollection tube and gently resuspended in Wash Buffer OW2 andre-centrifuged as describe herein.

The spin column was transferred to a new tube and eluted with 20 to 100μl of preheated (70° C.) Elution Buffer. The Oligotex resin was gentlyresuspended by pipetting up and down, and then centrifuged as above. Theelution procedure was repeated with fresh elution buffer. Otherwise iflow elution volume is necessary, the first eluate only may be used.

The absorbance was read, using diluted Elution Buffer as the blank.

Ethanol Precipitation

Before proceeding with cDNA synthesis, the mRNA was precipitated. Somecomponent leftover or in the Elution Buffer from the Oligotexpurification procedure will inhibit downstream enzymatic reactions ofthe mRNA.

0.4 vol. of 7.5 M NH₄OAc+2.5 vol. of cold 100% ethanol was added to theeluate. The solution was precipitated at −20° C. 1 hour to overnight (or20-30 min. at −70° C.). The precipitated solution was centrifuged at14,000-16,000×g for 30 minutes at 4° C. The pellet was washed with 0.5ml of 80% ethanol (−20° C.) then centrifuged at 14,000-16,000×g for 5minutes at room temperature. The 80% ethanol wash was repeated 1×. Thepellet was dried in the hood. (Do not speed vacuum). The pellet wassuspended in DEPC H₂O at 1 ug/μl concentration.

Cleaning Up Total RNA using Qiagen's RNeasy Kit

No more than 100 μg RNA should be added to an RNeasy column. The samplevolume was adjusted to 100 μl with RNase-free water, and 350 μl BufferRLT then 250 μl ethanol (100%) was added to the sample. The solution wasmixed by pipetting (do not centrifuge) then the sample applied to anRNeasy mini spin column. The mini spin column was centrifuged for 15 secat >10,000rpm. If concerned about yield, the flowthrough can bereapplied to the column and centrifuged again.

The column was transferred to a new 2-ml collection tube and 500 μl ofBuffer RPE was added and centrifuged for 15 sec at >10,000 rpm. Theflowthrough was discarded. 500 μl Buffer RPE was added to the mini-spincolumn again, and centrifuged for 15 sec at >10,000 rpm. The flowthroughwas again discarded, then centrifuged for 2 min at maximum speed to drycolumn membrane. The column was transferred to a new 1.5-ml collectiontube and 30-50 μl of RNase-free water was applied directly onto columnmembrane. The column was centrifuged for 1 min at >10,000rpm, and theelution repeated.

An absorbance reading was taken. If necessary, the eluate may beprecipitated with ammonium acetate and 2.5× volume 100% ethanol.

Making cDNA using Gibco's “SUPERSCRIPT® Choice System for cDNASynthesis” Kit

First Strand cDNA Synthesis

5 ug of total RNA or lug of polyA+ mRNA was used as starting material.For total RNA, 2 μl of SUPERSCRIPT® RT (kit with reverse transcriptasefor cDNA synthesis) was used (for polyA+ mRNA, use 1 μl of SUPERSCRIPT®RT). The final volume of the first strand synthesis mix should be 20 μl.RNA must be in a volume no greater than 10 μl. The RNA was incubatedwith 1 μl of 100 pmol T7-T24 oligo for 10 min at 70 C. On ice, 7 μl of:4 μl 5× 1^(st) Strand Buffer, 2 μl of 0.1M DTT, and 1 μl of 10 mM dNTPmix was added. The mixture was incubated at 37 C for 2 min, thenSUPERSCRIPT® RT was added.

The mixture was incubated at 37 ° C. for 1 hour.

Second Strand Synthesis

The 1⁴ strand reactions were placed on ice.

To the mixture was added:

91 μl DEPC H20

30 μl 5× 2^(nd) Strand Buffer

3 μl 10 mM dNTP mix

1 μl 10 U/μl E. coli DNA Ligase

4 μl 10 U/μl E.coli DNA Polymerase

1 μl 2 U/μl RNase H

The above should be made into a mix if there are more than 2 samples.The added mixture was incubated for 2 hours at 16 C.

2 μl T4 DNA Polymerase was added and further incubated for 5 min at 16C. 10 μl of 0.5M EDTA was added to stop the reaction.

Clean Up cDNA

The cDNA was cleaned up using Phenol:Chloroform:Isoamyl Alcohol(25:24:1) purification in gel tubes:

The PLG (phase lock gel) tubes were centrifuged for 30 sec at maximumspeed, and transferred to a new PLG tube. An equal volume ofphenol:chloroform:isamyl alcohol was added and shaken vigorously (do notvortex). The tubes were centrifuged for 5 minutes at maximum speed. Thetop aqueous layer solution was transferred to a new tube. The aqueoussolution was ethanol precipated by adding 7.5× 5M NH4Oac and 2.5× volumeof 100% ethanol. The tubes were centrifuged immediately at room temp.for 20 min, maximum speed. The supernatant was removed and the pelletwashed 2× with cold 80% ethanol. Remove as much ethanol wash as possiblethen let pellet air dry. The pellet was resuspended in 3 μl RNase-freewater.

In Vitro Transcription (IVT) and Labeling with Biotin

1.5 μl of cDNA was pipetted into a thin-wall PCR tube. NTP labeling mixwas added at room temperature to the PCR tube.

NTP Labeling Mix:

2 μl T7 10xATP (75 mM) (Ambion) 2 μl T7 10xGTP (75 mM) (Ambion) 1.5 μl  T7 10xCTP (75 mM) (Ambion) 1.5 μl   T7 10xUTP (75 mM) (Ambion) 3.75 μl  10 mM Bio-11-CTP 0.75 μl   10 mM Bio-16-UTP 2 μl 10x T7 transcriptionbuffer (Ambion) 2 μl 10x T7 enzyme mix (Ambion)

The final volume of the total reaction was 20 μl. The tubes wereincubated for 6 hours at 37° C. in a PCR machine.

RNeasy Clean-Up of IVT Product

See above for procedure.

The labeled cRNA is ethanol precipitated and resuspended in a volumecompatible with the fragmentation step.

Fragmentation

Approximately 15 ug of labeled RNA was fragmented using the followingtechnique. The fragmentation reaction volume was minimized toapproximately 10 μl volume, but not more than 20 μl due to magnesiumprecipitation problems with the hybridization buffer.

The RNA was fragmented by incubating at 94° C. for 35 minutes in 1×Fragmentation buffer.

5× Fragmentation Buffer:

200 mM Tris-acetate, pH 8.1

500 mM KOAc

150 mM MgOAc

The labeled RNA transcript was analyzed before and after fragmentation.Samples were heated to 65° C. for 15 minutes and electrophoresed on 1%agarose/TBE gels to get an approximate idea of the transcript size range

Microchip Array

The EOS HuO3 microchip array used in all experiments is a customizedAffymetric GENECHIP® oligonucleotide array comprising 59,680 probesetsrepresenting 46,000 unique sequences including both known and FGENESHpredicted exons that were based on the first draft of the human genome.The Hu03 probesets consist of perfect match probes only, most probesetshaving 6 or 7 probes.

Hybridization on Microchip Array

200 μl (10 ug cRNA) of hybridization mix was pipetted onto the chip. Ifmultiple hybridizations are to be done (such as cycling through a 5 chipset), then it is recommended that an initial hybridization mix of 300 μlor more be made.

Hybrization Mix: fragmented labeled RNA (50 ng/μl final conc.)

50 pM 948-b control oligo

1.5 pM BioB

5 pM BioC

25 pM BioD

100 pM CRE

0.1 mg/ml herring sperm DNA

0.5 mg/ml acetylated BSA

to 300 μl with 1× MES hybridization buffer

Hybridization signals were visualized using phycoerythrin-conjugatedstreptavidin.

Normalization of the Gene Expression Data

The gene expression data was normalized by fitting the probe-levelintensity data from each array to a fixed γ-distribution, using aninverse γ function to map the empirical cumulative distribution ofintensities to the desired γ distribution. This procedure is akin toother per-chip normalization procedures, such as fixing the mean and SDof each chip to a standard value, except it is more stringent in that itfixes the entire distribution of intensities rather than one or twoparameters. The purpose of per-chip normalization is to removebetween-chip variations, on the assumption that it is attributable tononbiological factors, i.e. technical noise. The scale parameter for thedistribution was chosen to yield a distribution with an arbitrary meanvalue of 300, and the shape parameter of 0.81 was chosen to reproducethe typical shape of the empirical distribution seen in good samples.

A single measure of average intensity was calculated for each probesetusing Tukey's trimean of the intensity of the constituent probes. Thetrimean is a measure of central tendency that is resistant to theeffects of outliers. Finally, a background subtraction was applied toeach average intensity measure to correct for nonspecific hybridization.The average intensity measure of a “null” probeset consisting of 491probesets with scrambled sequence was subtracted from all of the otherprobesetsw on the chip.

The instruction manuals for the products used herein are incorporatedherein in their entirety.

Labeling Protocol Provided Herein

Hybridization Reaction:

Start with non-biotinylated IVT (purified by RNeasy columns)

(see example 1 for steps from tissue to IVT)

IVT atisense RNA; 4 μg:   μl Random Hexamers (1 μg/μl): 4 μl H₂O:   μlTotal Volume: 14 μl 

-   -   Incubate 70° C., 10 min. Put on ice.

Reverse Transcription:

5X First Strand (BRL) buffer:   6 μl 0.1 M DTT:   3 μl 50X dNTP mix: 0.6μl H2O: 2.4 μl Cy3 or Cy5 dUTP (1 mM):   3 μl SS RT II (BRL):   1 μlTotal volume:  16 μl

-   -   Add to hybridization reaction.    -   Incubate 30 min., 42° C.    -   Add 1 μl SSII and let go for another hour.

Put on ice.

-   -   50× dNTP mix (25 mM of cold dATP, dCTP, and dGTP, 10 mM of dTTP:        25 μl each of 100 mM dATP, dCTP, and dGTP; 10 μl of 100 mM dTTP        to 15 μl H2O)

RNA Degradation:

-   -   Add 1.5 μl 1M NaOH/2 mM EDTA, incubate at 65° C., 10 min.

H₂O 86 μl 10N NaOH 10 μl 50 mM EDTA  4 μl

U-Con 30

500 μl TE/sample spin at 7000 g for 10 min, save flow through forpurification

Qiagen Purification:

-   -   suspend u-con recovered material in 500 μl buffer PB    -   proceed w/normal Qiagen protocol

DNAse Digest:

-   -   Add 1 μl of 1/100 dil of DNAse/30 μl Rx and incubate at 37° C.        for 15 min.    -   5 min 95° C. to denature enzyme

Sample Preparation:

-   -   Add:

Cot-1 DNA: 10 μl 50X dNTPs: 1 μl 20X SSC: 2.3 μl Na pyro phosphate: 7.5μl 10 mg/ml Herring sperm DNA 1 μl of 1/10 dilution Final Volume: 21.8μl

-   -   Dry down in speed vac.    -   Resuspend in 15 μl H₂0.    -   Add 0.38 μl 10% SDS.    -   Heat 95° C., 2 min.    -   Slow cool at room temp. for 20 min.

Put on slide and hybridize overnight at 64° C.

Washing after the hybridization:

3X SSC/0.03% SDS: 2 min. 37.5 mls 20X SSC + 0.75 mls 10% SDS in 250 mlsH₂O 1X SSC: 5 min. 12.5 mls 20X SSC in 250 mls H₂O 0.2X SSC: 5 min. 2.5mls 20X SSC in 250 mls H₂O

Dry slides in centrifuge, 1000 RPM, 1 min.

Scan at appropriate Photomultiplier Tube settings and fluorescencechannels.

CS1 is Over-Expressed in Leukocytes but not in Various Types ofNonlymphoid Tissues

To evaluate the expression profile of CS1, mRNA isolated from leukocytesand other tissues was analyzed by real-time PCR. As shown in FIG. 2A,the mRNA expression level was much higher in leukocytes than that ofmost other normal adult tissues. Other normal adult tissues that did notshow CS1 expression above baseline levels included adipose, adrenalgland, aorta, aortic valve, appendix, coronary artery, bladder, bone,bone marrow, breast, bronchus, cervix, brain, spinal cord, diaphragm,endometrium, epididymis, esophagus, gallbladder, ganglion, heart,larynx, lip, liver, lung, muscle, myometrium, vagus nerve, omentum, oralmucosa, ovary, pancreas, parathyroid, pharyngeal mucosa, placenta,prostate, retina, salivary gland, skin, stomach, synovium, testis,thymus, thyroid, tongue, trachea, umbilical cord, ureter,uterus, vagina,or vein. CS1 mRNA was expressed in selected samples of colon (2/11),kidney (1/20), small intestine (1/3), spleen, and tonsil (2/4). Theresults indicated that CS1 is primarily expressed in leukocytes andshould be a good target for autoimmune diseases.

CS1 Expression Increases in Multiple Activated Leukocyte Populations

To evaluate the correlation between the CS1 expression and activation ofleukocytes, CS1 mRNA expression was analyzed in multiple activated andnon-activated leukocytes populations. As shown in FIG. 2B, CS1expression increased in activated B cells, mature DC cells, activatedCD3 cells (low to moderate increase), activated CD4 cells (low levelincrease), and activated CD8 cells (low to moderate increase, dependingon donor) in comparison to their corresponding non-activated controlpopulations. These results indicated that CS1 over-expression correlateswith the activation of several leukocyte subsets.

Example 3 Production of Antigens for Generating Monoclonal AntibodiesAgainst CS1 Cloning:

The extracellular domain (ECD) of CS1 was isolated from Raji cells usingprimers flanking the extracellular domain of CS1 (CS1 ECD). The PCRproduct was gel purified and ligated into a vector encoding the constantregion of IgG3 (human Fc-γ3). The plasmid containing CS1 ECD-Fcγ3 waspurified on a large scale and confirmed by DNA sequencing.

CS1 ECD-Fcγ3 Stable Transfection:

50 μg of CS1 ECD-Fcγ3 plasmid was linearized with Fspl enzyme, and theDNA was precipitated in ethanol, washed, and resuspended in 500 μl ofsterile PBS. NSO cells were washed twice in cold PBS, and resuspended at2×10⁷ per one ml of PBS. An amount of 1×10⁷ cells was used fortransfection.

500 μl of NSO cells were combined with 500 μl of DNA in PBS. Cells wereelectroporated at 1.5V and 3 μF by a BioRad Gene pulser. Cells wereadded to 100 ml of DMEM complete media and plated into ten 96-wellplates. Mycophenolic selection media at 1 μg/ml was added 24 hours afterthe transfection. Positive transfectants were screened after day 10 andexpanded into 48- and 24-well plates. Positive transfectants werere-screened and high producers were expanded for protein purification.

Purification of CS1 ECD-Fcγ3 Protein:

Stable transfectants expressing the CS1-ECD Fcγ3 fusion protein wereexpanded into 600 ml of DMEM complete media with glucose additives forfive days. The fusion protein was purified on a protein G Sepharosecolumn and dialyzed against 1× PBS. Reduced and non-reduced forms of CS1ECD-Fcγ3 were analyzed by Coomassie. CS1 ECD Fcγ3 was also analyzed byWestern blot using anti-HuIgG, and confirmed by N-terminal sequencing.The purified CS1-Fc-γ3 fusion protein was used to immunize mice.

Production of CS1 ECD-myc-GPI Fusion Protein:

The extracellular domain (ECD) of CS1 was isolated from Raji cells usingprimers flanking the extracellular domain of CS1. The PCR product wasgel-purified and litigated into a vector expressing a myc tag andglycosyl phosphatidylinositol linkage for cell surface expression(myc-GPI vector). The plasmid containing CS1 ECD-myc-GPI was purified ona large scale and confirmed by DNA sequencing.

CS1 ECD-myc-GPI Stable Transfection:

50 μg of CS1 ECD-myc-GPI plasmid was linearized with Fsp1 enzyme, andthe DNA was precipitated in ethanol, washed, and resuspended in 500 μlof sterile PBS. NSO cells were washed twice in cold PBS and resuspendedat 2×10⁸ per one ml of PBS. An amount of 1×10⁸ cells was used fortransfection.

500 μl of NSO cells were combined with 500 μl of DNA in PBS. Cells wereelectroporated at 1.5 V and 3 μF by a Biorad Gene pulser. Cells weregrown in at 37° C. in 5% CO₂ with 1 μg/ml of mycophenolic selection, andwere later subcloned into 96-well plates. Positive transfectants werescreened with anti-myc antibody. High producers of CS1 ECD-myc-GPItransfectants were selected and expanded for in vitro assays.

Example 3 Production of Anti-CS1 Monoclonal Antibodies Immunogens forHuman CS1:

The purified recombinant human CS1 ECD-γ3 fusion protein was used toimmunize Balb/c mice via footpads (CS1 ECD refers to the extracellulardomain of CS1 described above). Briefly, mice were immunized in the hindfootpads with 10 μg protein with an equal volume of Ribi adjuvant in atotal volume of 25 μl. Footpad immunizations were performed 4 times at4- or 5-day intervals.

a. Cell Fusion:

Two mice immunized with CS1 ECD-γ3 were sacrificed. The poplitealfemoral and sacral lymph nodes were removed from the mice. Lymphocyteswere isolated from the tissues, and hybridomas were generated bystandard procedures. Briefly, hybridomas were generated by polyethyleneglycol (PEG) 1500 mediated fusion between lymphocytes and a murinemyeloma cell line (NSO cells). Fused cells were plated into 96-wellplates at a density of 10⁷ cells per plate. Selection of fused cells wasaccomplished using HAT (hypoxanthine, aminopterin, thymidine) media.

b. Screening of Hybridomas

Specificity of antibodies secreted by hybridomas was determined by aflow cytometry (FACS) based binding assay to CS1 expressing cells. FACSassay was performed using standard protocols. NSO stable transfectantsexpressing surface CS1 extracellular domain (2×10⁵) were resuspended in50 μl ice cold PBS with 50 μl hybridoma culture supernatant on ice for 1hour. After extensive washing, cells were incubated withphycoerythrin-conjugated goat anti-mouse IgG specific antibodies for 1hour on ice. Cells were washed again and cell-surface-bound antibodieswere detected by FACS using a Becton Dickinson FACScan. As shown inTable 1, the antibodies: Luc2, Luc3, Luc15, Luc20, Luc22, Luc23, Luc29,Luc32, Luc34, Luc35, Luc37, Luc38, Luc39, Luc56, Luc60, Luc63, or Luc90,bound strongly to the NSO-CS1 cells transfected with CS1, but not toNSO-FcRn. Anti-human CS1 antibodies bound to K562 and Daudi cells (whichare known to express native CS1) but not to negative control Jurkatcells. The data show that the produced anti-CS1 antibodies are capableof binding specifically to CS1. Also shown in the table were resultsfrom assaying (by ELISA) binding of Luc antibodies to CS1-γ3 fusionprotein versus negative control AR-G3 (γ3 fusion protein). Lucantibodies bound specifically to CS1-γ3 and not to the negative controlAR-γ3 fusion protein.

TABLE 1 Anti-human CS1 MABs generated from fusion 342 Results FACS (MFI)ELISA (capture) MAB NSO-CS1 NSO-FcRn K562 Daudi Jurkat CS1-G3 AR-G3Subclones Mouse Ig ISO 1 Luc2 162 <5 13.4 10.5 <5 1.0 0.2 Luc2-1 IgG1 2Luc3 377 <5 25.7 7.8 <5 0.9 0.5 Luc3-F IgG1, G2b 3 Luc15 110 <5 14.012.4 <5 1.1 0.3 Luc15-1 ND 4 Luc20 89 <5 8.0 12.6 <5 1.2 0.2 Luc20-1 ND5 Luc22 228 <5 14.7 6.1 <5 0.6 0.2 Luc22-1 IgG2b 6 Luc23 164 <5 19.610.2 <5 0.6 0.2 Luc23-1 IgG1 7 Luc29 86 <5 24.1 11.9 <5 0.9 0.2Luc29-D6, C8 IgG1 8 Luc32 201 <5 9.8 10.7 <5 0.8 0.2 Luc32-1 IgG2b 9Luc34 127 <5 26.2 10.3 <5 1.2 0.3 Luc34-1, 34-3 IgG1 10 Luc35 184 <510.6 29.7 <5 0.7 0.2 Luc35-1 IgG2a 11 Luc37 366 <5 12.8 7.2 <5 0.6 0.2Luc37-C12, F11 IgG2b 12 Luc38 112 <5 31.4 11.6 <5 0.8 0.2 Luc38-1 IgG2b13 Luc39 117 <5 12.0 17.5 <5 0.4 0.2 Luc39-E10 IgG2a 14 Luc56 132 <512.6 9.7 <5 1.0 0.2 Luc56-1 IgG2a 15 Luc60 230 <5 14.6 10.4 <5 0.9 0.3Luc60-2 IgG2b 16 Luc63 214 <5 15.8 12.7 <5 0.6 0.2 Luc63-1 IgG2a 17Luc90 237 <5 9.7 10.1 <5 0.8 0.1 Luc90-H1, D9 IgG2b ISO control 14 35.41 6.02 <5 0.16 0.14 Anti-Myc 193 335 6.62 6.85 <5 0.19 0.15

The Amino Acid Sequences of the Produced Anti-CS1 Monoclonal Antibodies

Antibody heavy and light chain variable regions were cloned usingstandard techniques. Briefly, total RNA from 1-5×10⁶ cells was used toprepare cDNA using a SMART RACE cDNA Amplification Kit (BD BiosciencesClontech) and variable regions were PCR amplified using gene specificprimers complementary to the mouse heavy and light chain constantregions.

The amino acid sequences of the mature heavy chain and the mature lightchain of the antibodies Luc90, Luc63, and Luc34 are shown in Table 4.

Example 4 Characterization of CS1 Antibodies

A flow cytometry competition assay was used to determine the epitopespecificity of 15 different anti-CS1 monoclonal antibodies. NSO stabletransfectants expressing surface CS1 (2×10⁵) were incubated on ice for 1hour with 50 μl anti-CS1 antibodies, including pairwise combinations ofLuc23, Luc29 Luc34, Luc35, Luc37, Luc38, Luc 63, and Luc 90. Inparallel, isotype control antibody (AIP-13) was used as a negativecontrol. Biotinylated anti-CS1 monoclonal antibodies (Luc23, Luc34,Luc37, Luc38, Luc63, and Luc90) were incubated at 1 μg/ml with thecell/antibody mixture for additional 30 minutes on ice. After extensivewashing, cells were incubated with phycoerythrin-conjugated streptavidinfor 1 hour on ice. Cells were washed and cell surface-bound biotinylatedantibodies were detected by FACS using a Becton Dickinson FACScan.

The unlabeled antibodies: Luc23, Luc34, Luc37, Luc38, Luc63, and Luc90were tested for the ability to compete with each other at aconcentration of 15 μg/ml, 3 μg/ml, and 0.6 μg/ml, and the competing orblocking antibodies were added at 1 μg/ml. AIP-13 was used as a negativecontrol, since this antibody does not bind CS1 or compete with any ofthe Luc antibodies. The level of flourescence (mean flourescenceintensity) (MFI) of the biotinylated antibodies is shown in the figure.A significant decrease in the MFI indicated competition for cell surfaceCS1 by biotinylated anti-CS1 by Mab versus unlabeled anti-CS1 Mab, by atleast 50% compared to the MFI of the control antibody.

FIG. 3 depicts an exemplary result of the competition assays between theLuc antibodies when the blocking Luc monoclonal antibodies were used ata concentration of 15 μg/ml and 3 mg/ml. The competition assaysindicated that several of the Luc antibodies contact distinct epitopes.Luc38 contacts an epitope distinct from the Luc37, 23, 90, and 63epitopes. Luc63 contacts a separate, non-overlapping epitope that isdistinct from the Luc37, 23, 90, and 38 epitopes. Luc90 contacts adifferent, non-overlapping epitope, distinct from the Luc 37, 23, 63,and 38 epitopes. Luc 23 contacts another non-overlapping epitope,distinct from the Luc90, 63,and 38 epitopes. Luc37 contacts anadditional non-overlapping epitope, distinct from the epitopes contactedby Luc90, 63, and 38. Luc63 contacts an overlapping epitope with Luc34,while Luc90 contacts an overlapping epitope with Luc34. Luc37 contactsan epitope that overlaps with the epitope of Luc23. Luc34 blocks orsignificantly decreases binding of all Luc antibodies, and may eithercontact a broad, exposed epitope or may have higher affinity for CS1.Luc37, Luc23, and Luc38 do not block binding to CS1 by the Luc34antibody. Epitopes for Luc37, Luc23, and Luc38 may be “buried” withinthe CS1 secondary structure, or the affinity for CS1 may be lower thanthe affinity of the Luc34 antibody.

The relative affinities of three monoclonal antibodies were also testedby Biacore analysis. Kinetic Analysis of CS1 MAbs by SPRKineticsmeasurements between human CS1-Fc fusion protein and anti-human CS1monoclonal mouse antibodies Luc34.1, 63.2, and 90H1 were performed usingBIAcore 2000 (BIAcore, Sweden). Regeneration condition was establishedby immobilizing over 10 000 RUs of each antibody onto different flowcells and injecting CS1-Fc over the surface, followed by testing aseries of different buffers until the best one was found to optimize theclearance of CS1-Fc from each antibody. A buffer of 10 mM Glycine, pH2.0was found to be the optimal regeneration buffer and was immediatelytested for its reproducibility over 10 cycles of CS1-Fc injection andbuffer regeneration. The buffer was found to be suitable forregenerating the antibody surface reproducibly. Hence 10 mM Glycine,pH2.0 was the designated regeneration buffer for the CS1-Fc and antibodyBIAcore experiments.

CS1 antibody produced in-house was immobilized with low response units(RUs) ranging from 99.4 RUs to 133.7 RUs on the Research-grade CMSsensor chip by the BIAcore amine coupling reagents(N-ethyl-N′-dimethylaminopropylcarbodiimide, EDC; N-hydroxysuccinimide,NHS; and ethanolamine HCl, pH 8.5). Assays were run at a flow rate of 30ul/min at room temperature. A three-minute association phase of CS1-Fcwas followed by ten-minute injection of running buffer (10 mM Hepes, 300mM sodium chloride, 3 mM EDTA, 0.05% P-20, pH7.4) to monitordissociation for each binding cycle, with different CS1-Fcconcentrations per cycle. The regeneration surface was regenerated with10 mM Glycine, pH2.0. The binding kinetics of each CS1-Fc and antibodypair was calculated from a global analysis of senorgram data collectedfrom twelve different concentrations of CS1-Fc (1024 nM, 512 nM, 256 nM,128 nM, 64 nM, 32 nM, 16 nM, 8 nM, 4 nM, 2 nM, 1 nM, 0.5 nM) induplicate, using the BIAevaluate program. Double referencing was appliedin each analysis to eliminate background responses from referencesurface and buffer only control. The affinity (K_(D)) of binding wasobtained by simultaneously fitting the association and dissociationphases of the sensorgram from the analyte concentration series using thebivalent analyte model from BIAevaluate software. The experiment wasperformed three times to study the standard deviation of the data.

The binding affinities of Luc 90.H1, Luc63.2, and Luc43.1 are summarizedin FIG. 4. Luc90.H1 has highest binding affinity among these threeantibodies. The binding affinity of Luc90.H1 is 5.5 fold higher thanthat of Luc 63.2 and 28 fold higher than that of Luc34.1.

Immunohistological Staining with Anti-CS1 Antibodies:

The CS1-transfected cells were also examined for immunohistologicalstaining with anti-CS1 antibodies. An amount of 10 μg/ml of primarymonoclonal anti-CS1 antibody was added to the cells transfected withCS1. The cells were then blocked with serum and incubated with thebiotin-anti-mouse-Ig. Avidin-peroxidase was then mixed with the cellsand developed with AEC (a standard peroxidase reagent). The red color ofAEC indicated the positive staining while the nuclei of the tested cellswere counter-stained with hematoxylin (blue). The data indicated thatCS1-transfected cells were positively stained with the anti-CS1antibodies, showing that the produced anti-CS1 antibodies are capable ofbinding to CS1 expressed on the cell surface (FIG. 5A). Thus, theanti-CS1 antibodies are suitable for use not only in detectingexpression on the surface of peripheral blood cells in solution, butalso in detecting by immunohistochemistry (IHC), which is typically usedto analyze tissue sections (for example, patient lymph nodes or tissuebiopsies).

FIG. 5B shows immunohistological staining of inflamed tonsil with twoanti-CS1 antibodies, Luc90 and Luc63. Panels C and D in FIG. 5B showstaining with CD138 which stains plasma cells and epithelial cells. Theupper panels (FIG. 5B, panels A and B) show serial section staining withanti-CS1 antibodies. From the overlapping pattern of the staining, it isevident that CS1 antibodies stain plasma cells in inflamed tonsil.

FIG. 5C shows immunohistological staining of synovial tissue from thejoint of a patient with rheumatoid arthritis with anti-CS1 Luc63. Plasmacells have infiltrated in the synovium as seen by the staining withCD138 (right hand, top panel). From the overlapping pattern of thestaining (compare right hand top panel to left hand top panel), it isevident that anti-CS1 antibodies stain plasma cells in the joints ofpatients with rheumatoid arthritis.

CS1 Protein Expression Pattern:

The CS1 protein expression was further examined with the produced Lucantibodies through FACS analysis (FIG. 6). PBMCs were isolated fromhealthy individuals and from lupus patients by a standard Ficoll Hypaquegradient centrifugation procedure. PBMCs were stained with antibodies asindicated following standard procedures. For pokeweed mitogen (PWM)activation of PBMCs, PWM was added at 1:100 dilution to PBMCs, whichwere subsequently placed at 37° C. in 7% CO₂ for 8 days. PWM-stimulatedcells were harvested and washed prior to antibody staining. The mouseanti-CS1 antibodies used herein are Luc90 (IgG₂b), Luc63, Luc38 andother produced anti-CS1 Luc antibodies. Isotype control antibodies wereisotype matched mouse IgG antibodies.

The results indicated that CS1 was positively expressed on activated Bcells, CD8⁺ T cells (both activated and naïve), NK cells (CD3-CD56⁺),NKT cells (CD56⁺CD3⁺), CD14^(+/lo) leukocytes (monocytes and/ormacrophages), and CD4⁺ T cells (low level on in vitro activated cells).CSI was expressed on these cell populations from both healthy adults andlupus patients. No significant CS1 protein expression was detected onunactivated CD4⁺ T cells from healthy adults, platelets, HuVECs, kidneycells, bronchial airway cells, small airway cells, prostate cells, livercells, and breast cells.

Sample staining of activated B cells is shown in FIG. 6, where stainingof PWM-activated PBMCs is shown as the heavy line, while isotype controlstaining and unactivated PBMCs was shown as the underlaid dotted lines.The CS1 expression pattern is significant, because a therapeuticantibody ideally binds primarily to target cells and does not bind toother cells and tissues, especially platelets. The data suggest anti-CS1antibodies are suitable candidate therapeutic antibodies.

Example 5 Humanization of CS1 Antibodies

This example describes the humanization of the murine anti-CS1monoclonal antibody Luc63 (MuLuc63). Humanization of MuLuc63 was carriedout essentially according to the procedure of Queen, C. et al. (Proc.Natl. Acad. Sci. USA 86: 10029-10033 (1989)). First, human VH and VLsegments with high homology to the MuLuc63 VH and VL amino acidsequences, respectively, were identified. Next, the CDR sequencestogether with framework amino acids important for maintaining thestructures of the CDRs were grafted into the selected human frameworksequences. The resulting humanized monoclonal antibody (HuLuc63) wasexpressed in the mouse myeloma cell line NSO. The humanized HuLuc63antibody bound to recombinant human CS1 in an ELISA assay with an EC50value of 70.1 ng/ml, similar to the EC50 value of 66.1 ng/ml determinedfor MuLuc63 in the same assay, indicating that HuLuc63 retained highbinding affinity for human CS1.

Cloning and Sequencing of MuLuc63 Variable Region cDNAs

Total RNA was extracted from approximately 5×10⁷ hybridoma cellsproducing MuLuc63 using TRIzol reagent (Life Technologies, Inc.,Rockville, Md.). Double-stranded cDNA was synthesized using the SMARTRACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, Calif.)following the supplier's protocol. The variable region cDNAs for theheavy and light chains were amplified by polymerase chain reaction (PCR)using 3′ primers that anneal respectively to the mouse gamma and kappachain C regions, and a 5′ universal primer provided in the SMART RACEcDNA Amplification Kit. For VH PCR, the 3′ primer has the sequence5′-AGCTGGGAAGGTGTGCACAC-3′ (SEQ ID NO:51). For VL PCR, the 3′ primer hasthe sequence 5′-TTCACTGCCATCAATCTTCC-3′ (SEQ ID NO:52). The VH and VLcDNAs were subcloned into the pCR4Blunt-TOPO vector (InvitrogenCorporation, Carlsbad, Calif.) for sequence determination. DNAsequencing was carried out by PCR cycle sequencing reactions withfluorescent dideoxy chain terminators (Applied Biosystems, Foster City,Calif.) according to the manufacturer's instructions.

Four plasmid clones were sequenced for each of the heavy and lightchains. Unique sequences homologous to typical mouse heavy and lightchain variable regions were identified. The cDNA sequences along withdeduced amino acid sequences of the heavy and light chain V regions ofMuLuc63 are shown in Tables 5 and 6.

Design of HuLuc63 V Regions

Humanization of the antibody V regions was carried out as outlined byQueen, C. et al. (Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989)).First, a molecular model of the MuLuc63 variable regions was constructedwith the aid of the computer programs ABMOD and ENCAD (Levitt, M., J.Mol. Biol. 168: 595-620 (1983)). Next, based on a homology searchagainst human antibody cDNA sequences, the human VH sequence E55 3-14(Cuisinier et al, Eur. J. Imm. 23:110-118 (1993)) and the J segment JH1(Ravetch, J. V. et al., Cell 27: 583-591 (1981)) were selected toprovide the frameworks for the HuLuc63 heavy chain variable region. Forthe HuLuc63 light chain variable region, the cDNA VL sequence III-2R(Manheimer-Lory et al, J. Exp. Med. 174:1639-1652 (1991)) was used. Theidentity of the framework amino acids between MuLuc63 VH and theacceptor human frameworks was 81.6% (71/87), while the identity betweenMuLuc63 VL and the acceptor human frameworks was 76.3% (61/80).

At framework positions in which the computer model suggested significantcontact with the CDRs, the amino acids from the MuLuc63 V regions weresubstituted for the original human framework amino acids. This was doneat residues 28, 48, 49, 66 and 68 of the heavy chain (Table 7). For thelight chain, replacement was made at residue 60 (Table 8). Note that thenumbering system used here is that of Kabat (Sequences of Proteins ofImmunological Interest, 5th ed., National Institutes of Health,Bethesda, Md. (1991)).

In addition, inspection of the MuLuc63 amino acid sequence revealed asite for potential N-linked glycosylation in CDR2 of the VH region. SuchN-linked glycosylation sites have the general sequence N-X-T/S (whereN=asparagine, X=any amino acid, and S/T=serine or threonine). Since thepresence of N-linked glycosylation in the variable domain could haveundesirable effects during development of HuLuc63 as a therapeuticantibody, the potential glycosylation site in CDR2 (N-Y-T) waseliminated by substitution of threonine with alanine mutation (N-Y-A) inthe humanized design.

The alignments of MuLuc63, HuLuc63, and the human acceptor amino acidsequences for VH and VL are shown in Tables 7 and 8, respectively.

Construction of HuLuc63 VH and VL Genes

A gene encoding each of HuLuc63 VH and VL was designed as a mini-exonincluding a signal peptide, a splice donor signal, and appropriaterestriction enzyme sites for subsequent cloning into a mammalianexpression vector. The splice donor signals in the VH and VL mini-exonswere derived from the corresponding human germline JH6 and JK4sequences, respectively. The signal peptide sequences in the HuLuc63 VHand VL mini-exons were derived from the corresponding MuLuc63 VH and VLsequences, respectively. The nucleotide sequences of Luc63 VH and VLgenes along with deduced amino acid sequences are shown in Tables 5 and6.

The HuLuc63 VH and VL genes were constructed by extension of overlappingsynthetic oligonucleotides ranging in length from 33 to 43 bases and PCRamplification. (Stemmer et al, Gene 164:49-53 (1995)). Oligonucleotidesfor the synthesis of HuLuc63 VH and VL genes are listed in Table 9.

The PCR-amplified fragments were purified by Qiaquick PCR purificationkit (Qiagen) and digested with MluI and XbaI. The HuLuc63 VH gene wassubcloned into pHuHCg1.D to create plasmid pHuHCg1.D-HuLuc63. TheHuLuc63 VL gene was subcloned into pHuCkappa.rgpt.dE, a derivative ofthe kappa light chain expression vector pOKT3.Vk.rg (Cole, M. S. et al.,J. Immunol. 159: 3613-3621 (1997)), to create plasmidpHuCkappa.rgpt.dE-HuLuc63.

Expression of HuLuc63

HuLuc63 IgG1/κ antibody was produced by transient transfection of tissueculture cells. Human embryonic kidney cell line 293-H (Invitrogen,Carlsbad, Calif.) was maintained in DMEM (BioWhittaker, Walkersville,Md.) containing 10% FBS (HyClone, Logan, Utah) and non-essential aminoacids (Invitrogen). 293-H cells were plated at 1×10⁶ cells per well in avolume of 2.5 ml in a 6-well plate the day before transfection usingregular media (DMEM+10% FBS +non-essential amino acids). On the day oftransfection, 4 μg of plasmid DNA per well was diluted in 250 μl ofHybridoma-SFM (H-SFM, Invitrogen). 10 μl of lipofectamine 2000 Reagent(LF2000, Invitrogen) per well was diluted in 250 μl H-SFM. Diluted DNAwas combined with diluted LF2000 and incubated for 20 minutes to allowDNA-LF2000 complexes to form. 500 μl of DNA-LF2000 complexes were addedto each well and mixed by tilting the plate back and forth. Cells wereincubated for 5 days before harvesting supernatant for analysis.

Expression of HuLuc63 was measured by sandwich ELISA. Immulon 4 HBXplates (Thermo Labsystems, Franklin, Mass.) were coated overnight at 4°C. with 100 μl/well of 1.8 μg/ml of goat anti-human IgG Fcγ-chainspecific polyclonal antibodies (Jackson ImmunoResearch Laboratories,Inc., West Grove, Pa.) in 0.2 M sodium carbonate-bicarbonate buffer, pH9.4, washed with Wash Buffer (PBS containing 0.1% Tween 20), and blockedfor 30 min at room temperature with 150 μl/well of SuperBlock BlockingBuffer in TBS (Pierce Chemical Company, Rockford, Ill.). After washingwith Wash Buffer, samples containing HuLuc63 were appropriately dilutedin ELISA Buffer (PBS containing 1% BSA and 0.1% Tween 20) and 100μl/well was applied to the ELISA plates. As a standard, humanizedanti-CD33 IgG1/κ monoclonal antibody HuM195 (Co, M. S. et al., J.Immunol., 148: 1149-1154 (1992)) was used. After incubating the platesfor 1 hr at room temperature and washing with Wash Buffer, boundantibodies were detected using 100 μl/well of a 1:1000 dilution ofHRP-conjugated goat anti-human kappa chain polyclonal antibodies(SouthernBiotech, Birmingham, Ala.). After incubating for 1 hr at roomtemperature and washing with Wash Buffer, color development wasperformed by adding 100 μl/well of ABTS substrate (KPL, Inc.,Gaithersburg, Md.). Color development was stopped by adding 100 μl/wellof 2% oxalic acid. Absorbance was read at 415 nm using a VersaMaxmicroplate reader (Molecular Devices Corporation, Sunnyvale, Calif.).

Binding Properties of MuLuc63 and HuLuc63

The affinities of MuLuc63 and HuLuc63 to human CS-1 were analyzed bydirect binding ELISA. Wells of 96-well ELISA plates (Immulon 4 HBXplates, Thermo Labsystems, Franklin, Mass.) were coated with 100 μl of 1μg/ml soluble human CS1-human Fcγ3 fusion protein in PBS overnight atroom temperature. After washing with Washing Buffer, wells were blockedwith 150 μl of Superblock Blocking Buffer for 30 minutes at roomtemperature. Transiently expressed HuLuc63 antibody or purified MuLuc63antibody were appropriately diluted in ELISA Buffer and applied to ELISAplates (100 μl per well). ELISA plates were incubated for 1 hour at roomtemperature and the wells were washed with Washing Buffer. Then 100 μlof HRP-conjugated goat-anti-human Cκ antibody or HRP-conjugatedgoat-anti-mouse Cκ antibody (both from Southern Biotech) diluted 1:1000in ELISA Buffer was added to each well of the HuLuC63 and MuLuc63plates, respectively and incubated at room temperature for 1 hour. Afterwashing with Washing Buffer, 100 μl of ABTS substrate (KPL) was added toeach well. Color development was stopped by adding 100 μl of 2% oxalicacid per well. Absorbance was read at 415 nm using a VERSAmax microplatereader. The results of the ELISA binding experiments are shown in FIG.7. MuLuc63 and HuLuc63 bind to human CS-1-Fcγ3 in aconcentration-dependent manner. The E₅₀ value of HuLuc63, obtained usingthe computer software GraphPad Prism (GraphPad Software Inc., San Diego,Calif.), was 70.1 ng/ml. This is similar to the EC50 value of 66.1 ng/mlobtained for muLuc63, indicating that humanization of mouse anti-CS1monoclonal antibody MuLuc63 was successful: HuLuc63 retained highbinding affinity to human CS1. A model of the humanized Luc63 variableregion is shown in FIG. 8.

Example 6 Role of CS1 in Autoimmune Disorders CS1 is Highly Expressed inStimulated T and B Cells, as Compared to Unstimulated Cells:

To determine the expression of CS1, an in-vitro assay was set up tostimulate peripheral blood B and T lymphocytes, using pokeweed mitogen(PWM) and phytohemagglutinin (PHA) stimulants. Unstimulated controlperipheral blood mononuclear cells were prepared in parallel with nostimulation. PolyA⁺ mRNA was isolated and cDNA was synthesized fromthese samples using standard techniques. The CS1 gene was amplified byPCR using CS1-specific oligonucleotide primers (see above) andexpression was quantified using Biorad Gel Doc 2000. Signal intensitieswere normalized to control human β-actin. Real time PCR analysisindicated that CS1 showed about 23-fold up-regulation in activatedperipheral blood B cells and about 30-fold up-regulation in activatedperipheral blood T lymphocytes, as compared to unstimulated cells (FIG.9).

CS1 is Up-Regulated in the Lupus Patient's Peripheral Blood BLymphocytes as Compared to those of the Age-Matched Healthy Adults:

To evaluate CS1 expression in lupus patients compared to healthyindividuals, peripheral blood B lymphocytes were isolated by cellsorting of CD19⁺ cells from a lupus patient versus a pool of healthyadults. PolyA⁺ mRNA was isolated and cDNA was synthesized by usingstandard techniques. CS1 expression was evaluated by real time PCR usingoligonucleotide primers specific to CS1. Real time PCR data indicatedthat CS1 is up-regulated about 2-fold in B lymphocytes from the lupuspatient as compared to the healthy individuals. Upon normalization withβ-actin, the CS1 gene was increased 2.3 fold in the lupus patient's Blymphocyte cDNA as compared to the healthy individuals' cDNA. Whennormalized with 18S rRNA primers, CS1 was increased 1.8 fold in therespective cDNA samples (FIG. 10).

Up-Regulation of Mouse Novel Ly9 in Activated B and Activated T Cells:

Mouse novel Ly9 is a proposed orthologue of human CS1 (Tovar et al.,Immunogenetics 54: 394-402 (2002)). The expression of mouse novel Ly9 inactivated B and activated T cells was examined with real time PCR. Thedata showed that mouse novel Ly9 is up-regulated in activated B andactivated T cells.

Mouse novel Ly9 expression was analyzed with an ABI GeneAmp 5700Sequence Detection System (see Example 2). Upon normalization with 18SrRNA primers, the Ly9 gene was increased by 3 fold in theconA-stimulated cDNA, and up-regulated by 6 fold in LPS-stimulated cDNAas compared to the unstimulated splenic cDNA.

Upregulation of CS1 in Inflammatory Bowel Disease Tissue

The expression of IBD modulator protein(s) in IBD tissue (both Crohn'sdisease and ulcerative colitis) versus normal tissue was determined onmicrochip arrays as described above. Oligonucleotide microarrays wereinterrogated with cRNAs derived from multiple tissues. Morespecifically, cRNAs were generated by in vitro transcription assays(IVTs) from nine IBD and nine matched adjacent normal bowel specimens,and 24 colonic epithelial samples. cRNA hybridization to theoligonucleotide microarrays was measured by the average fluorescenceintensity (AI), which is directly proportional to the expression levelof the gene.

The data was analyzed by comparing gene expression levels in IBD tonon-pathogenic adult tissues and organs. One of the genes identifiedwith a significant increase in gene expression in inflammatory boweldisease tissue compared to normal tissue is CS1. FIG. 11 is a graphicrepresentation of the microarray analysis, showing that CS1 geneexpression is increased in ulcerative colitis and Crohn's as compared tohealthy adult colonic epithelial cells.

To further evaluate CS1 expression in inflammatory bowel diseasepatients compared to healthy individuals, samples from diseased sectionsof large intestine from 2 Crohn's disease patients and 3 ulcerativecolitis patients versus normal large intestine samples from 3 healthyadults were disaggregated, washed, and placed in TRIZOL®. Total RNA wasisolated following the manufacturer's protocol. The total RNA wastreated with Rnase free Dnase (GenHunter). The Dnase digested RNA wasextracted with phenol/chloroform, and precipitated overnight withethanol. RNA was washed with 75% ethanol and dissolved in nuclease-freewater. RNA was quantified and the integrity of the RNA was analyzed onan agarose gel. Real time PCR data (FIG. 12) indicated that CS1 isup-regulated 7-fold and 6-fold in diseased large intestine from Crohn'spatients (n=2) and 13-fold, 14-fold and 46-fold in diseased largeintestine from ulcerative colitis patients (n=3) compared to poolednormal intestine from healthy individuals (n=3).

Example 7 CS1 Expression on Cancer Cells CS1 Protein Expression Pattern:

CS1 protein expression was further examined with the produced Lucantibodies through FACS analysis. Cell lines were incubated withanti-CS1 Luc90.H1 antibodies or mouse IgG2b isotype control antibodiesfor 30 minutes on ice. Cells were washed with PBS and phycoerythrin(PE)-conjugated anti-mouse Ig was added to the cells and incubated for30 minutes on ice. Cells were washed and analyzed by flow cytometry on aFACS Caliber (Becton Dickinson). Histogram plots are shown in FIG. 13,where signal from Luc90.H1 antibodies is shown as the overlapping boldline. Underlying lines include negative controls (unstained cells,secondary antibody (anti-mouse Ig-PE with no primary antibody), orisotype control antibody.). These data show that CS1 is expressed inARH-77 leukemia line cells, CESS and IM9 B lymphoblastoid cell lines,and L363, LP1, and OPM2 myeloma cell lines.

Samples from patients with multiple myeloma (n=21 bone marrow samples),a patients—with MGUS (monoclonal gammopathy of unknown significance;n=1), a patient with plasma cell leukemia (n=1), CD34+ stem cellsmobilized from bone marrow (n=5), normal marrow cells (n=3), normallymph node tissue (n=1), patients with Chronic Lymphoblastic Leukemia(CLL; n=15), patients with acute myelogenous leukemia (AML; n=11), apatient with non-Hodgkin's lymphoma (NHL; n=1), and a patient withHodgkin's lymphoma (n=1) were incubated with FITC conjugated antibodiesto CS1 (Luc90 or Luc63), CD45-PerCP, CD38-PE, and/or CD138-PE andprocessed as detailed above for FACS analysis of myeloma cells (see FIG.14). The mouse anti-CS1 antibodies used herein are Luc90 (IgG₂b), Luc63(IgG2a), Luc38 (IgG2b) and other produced anti-CS1 Luc antibodies.Isotype control antibodies were isotype matched mouse IgG antibodies.

Bone marrow aspirates were obtained from multiple myeloma patients fromthe Cleveland Clinic. Myeloma cell lines (LP1, L363, OPM2, NCI-H929,RPMI 8226, and U266 B1), the leukemia cell line ARH-77, B lymphoblastoidlines (IM9, CESS), and bone marrow cells were stained with anti-CS1monoclonal antibodies versus isotype control antibodies (BectonDickinson) following a standard staining protocol. Cells were washed,placed in staining buffer (RPMI, 10% FBS for human cells or DMEM, 10%FBS), and anti-CS1 versus isotype control antibodies were added at 0.5-1ug antibody per million cells in 0.1 ml final volume. For patientsamples, red blood cells were lysed, and cells were pelleted in acentrifuge and resuspended in staining buffer. For antibodies that werenot directly conjugated to FITC, second stage antibodies were added at0.5-1 ug antibody per million cells in 0.1 ml final volume. Cells werewashed and resuspended in staining buffer for FACS analysis on a BectonDickinson FACSCaliber using CellQuest software. To distinguish plasmacells, multiple myeloma bone marrow cells were stained with anti-CD45,anti-syndecan-1 (CD138), and anti-CD38 monoclonal antibodies.Anti-syndecan-1 (CD138) specifically stains plasma cells and not otherleukocytes.

The results show that CS1 is highly expressed on plasma cells (eg CD138+cells) from multiple myeloma patients (FIGS. 14A-14H), plasma cells froma plasma cell leukemia patient (FIG. 14I), and on several myeloma celllines (L363, LP1, and OPM2; see FIG. 13). A total of 21 different bonemarrow samples from multiple myeloma patients have been assayed by flowand for all 21 out of 21 samples, virtually all of the bone marrowplasma cells express CS1. CS1 is also expressed on ARH-77 leukemia cellsand B lymphoblastoid cell lines (IM9 and CESS) (see FIG. 13).

Example 8 Expression of CS1 on Plasma Cells from Myeloma Patient

Bone marrow samples from a multiple myeloma patient were stained withCD138-PE, CD45PerCP, Luc90-FITC, and/or IgG2b-FITC (isotype controlantibody) and analyzed by FACS as detailed above (see Example 5). Gatedcells are as follows: gate R1 contains lymphocytes (“R1”), gate R2contains monocytes (“R2”), gate R3 contains granulocytes (“R3”), gate R4contains erythroid cells (“R4”), gate R5 contains plasma cells (“R5”),and gate R6 contains blasts (“R6”). FIG. 15 shows that CS1 is expressedon plasma cells (eg CD138+ cells) from the multiple myeloma patient.

Example 9 Anti-CS1 Monoclonal Antibody Decreases IgM Secretion byActivated Peripheral Blood B Cells

Peripheral blood mononuclear cells from a normal adult were isolated bya standard Ficoll gradient, incubated with pokeweed mitogen at 10 μg/ml(GIBCO/BRL, England, the United Kingdom), and plated in a 24-well platein a total volume of 1 ml. Monoclonal antibody (mouse anti-human CS1(Luc63) or mouse IgG isotype control) was added to sample wells at 100μg/ml or 10 μg/ml. The cells and the antibody were incubated at 37° C.in 7% CO₂ for 8 days. Supernatants from cultures were isolated and IgMwas assayed by ELISA as described above. As shown in FIG. 16, theantibody Luc63 at 100 μg/ml or 10 μg/ml (PwLuc100 and PwLuc10,respectively) decreased the secretion of IgM of the peripheral bloodmononuclear cells compared to IgM secretion by cells incubated with theisotype control at 100 μg/ml or 10 μg/ml(PwIg100 and PwIg10,respectively) or no antibody (Pw(−)).

Anti-CS1 Monoclonal Antibody Decreases IgM Secretion by Auto-ImmuneDisease Patient Activated Peripheral Blood B Cells:

Supernatants from the cell cultures of peripheral blood mononuclearcells were isolated as detailed above and assayed by ELISA. Immulon-1plates were coated with 100 μl of 1 μg/ml mouse anti-human IgMmonoclonal antibody (catalog #05-4900, Zymed Laboratories, Inc., SouthSan Francisco, Calif.) in PBS. The plates were blocked for 1 hour withELISA Buffer (‘EB’=PBS+0.1% BSA+0.05% Tween 20). The culturesupernatants were added at various dilutions (in EB) at 100 μl/well. Thesupernatants and standard human IgM (catalog #009-000-012, JacksonLaboratory, Bar Harbor, Me.) were incubated for 1-2 hours at roomtemperature. Captured human IgM was developed with goat anti-humanIgM-HRP polyclonal antibody (catalog #2020-05, Southern BiotechAssociation, Birmingham, Ala.) and HRP substrate, by following themanufacturer's protocol. Bound IgM was visualized by spectrophotometry(405 nm OD) on a standard ELISA plate reader. As shown in FIG. 17, theamount of the secreted IgM of the lupus patient PBMCs was reduced by thetreatment with anti-CS1 antibodies (Luc90H1) as compared to the isotypecontrol. A positive control anti-CD2 antibody (GLO1) showed thatanti-CS1 is even more robust at reducing IgM production than theanti-CD2 antibody.

Anti-CS1 Monoclonal Antibody Decreases IgG Production by PeripheralBlood B Cells from Healthy Adults and from Auto-Immune Disease Patients

IgG production by peripheral blood B cells from healthy adults andautoimmune disease (lupus) patients were analyzed the same way as theIgM production. As shown in FIG. 18, the total production by healthyadult peripheral blood mononuclear cells 9 days after the treatment withthe anti-CS1 antibody (Luc90H.1) decreased by about 23% as compared withthe IgG2b isotype control. The total production of IgG by lupus patientperipheral blood mononuclear cells 9 days after the treatment withanti-CS1 antibody (Luc90H.1) decreased by about 56% as compared with theIgG2b isotype control. Tables 3A and B summarize the inhibition of theIgG production by a number of generated anti-CS1 antibodies. As shown inTable 3A, Luc90.H1 reduced by about 40% the IgG production by PBMCsactivated with lipopolysaccharide or pokeweed mitogen. Luc34.1 reducedby about 38% the IgG production by PBMCs activated with pokeweedmitogen. As shown in Table 3B, Luc 90.H1 reduced the IgG production ofPBMCs of a healthy adult and a mature B cell line (IM9 cells) by about48%. Luc 34.1 reduced the IgG production of PBMCs of the healthy adultby about 53%. Luc 63.2 reduced the IgG production of PBMCs and IM9 cellsby about 47%. From these experiments, it is evident that Luc 90H.1,Luc34.1, and Luc 63.2 are the best functional antibodies. From epitopemapping, Luc90 and Luc63 have nonoverlapping epitopes.

TABLE 3A ANTI-CS-1 DECREASES IG PRODUCTION By In Vitro Activated B CellsMean Percent Decrease Compared to Isotype Control In Vitro ActivatedANTI-CS-1 Average % Decrease PBMCs MAB HuIgG ± SE Lipopolysaccharide Luc90.H1 41% ± 8% (n = 3) Pokeweed Mitogen Luc 90.H1 39% ± 9% (n = 4)Pokeweed Mitogen Luc 34.1 38% ± 7% (n = 4)

TABLE 3B Summary of Ig Production Assays with Anti-CS-1 Antibody PanelMEAN PERCENT CHANGE IN IG COMPARED TO ISOTYPE CONTROL ANTI-CS1 PMBC PMBCAverage % Mab Donor 55 Donor 705 IM9 Change in Ig Luc90H.1 −44% −56%−43% −48% Luc37 +11% −43% −11% −14% Luc23 −13% −4% +6% −4% Luc63.2 −55%−51% −36% −47% Luc34.1 −64% −49% −45% −53% Luc38.1 −22% −44% −21% −29%Luc29D6 −43% −44% −25% −37% Relative Decrease in Ig Production: Group A(>45% dec): Luc 90, 63, 34 GROUP B (29-37% DEC): LUC38, 29D6 GROUP C(4-14% DEC): LUC 37, 23

The experimental results indicated that anti-CS1 antibodies decrease theproduction of both IgG and IgM by peripheral blood B cells in vitro.

Example 10 In Vivo Reduction of IgG by CS1 Monoclonal Antibodies in aSCID-HuPBMC Mouse Model SCID-HuPBMC Mouse Model

Human peripheral blood mononuclear cells (PBMCs) were isolated bystandard Ficoll-paque (Amersham Biosciences) density gradients andresuspended in phosphate buffered solution (PBS) at 2×10⁷ PBMCs/ml.Resuspended PBMCs (1 ml) were injected intraperitoneally (i.p.) intoC.B-17 SCID mice. Two to three weeks after PBMC injection, serum sampleswere drawn from mice and assayed for human IgG by ELISA. Engrafted mice(producing >1 μg/ml human IgG in serum) were randomized into treatmentgroups and then treated with mouse anti-human CS-1 monoclonal antibodies(Luc90.H1 or Luc63.2.22), mouse isotype control antibodies (IgG2b orIgG2a, respectively), or PBS. Mice were dosed with 200 ug of antibody in500 μl PBS every 3-4 days with 3 or 4 doses of antibody. Mouse serum wasanalyzed for human IgG by ELISA using standard protocols.

The percent change in serum human IgG was calculated for each mouse bysubtracting human IgG concentration prior to the first dose of antibody(day 0) from the human IgG concentration post dose (day x), dividing bythe human IgG concentration prior to the first dose (day 0), andmultiplying by 100, e.g., [(day x−day 0)/day 0]×100. Data are shown asthe average percent change with the standard error for each group ofmice. Human IgG concentrations are the average concentration with thestandard error for each group of mice. The Welch 2 sample t-test wasused to compare the percent change in human IgG across treatment groups.

Anti-CS1 Antibodies Reduced the Production of Human IgG In Vivo

The data shows that anti-CS1 antibodies of the present invention reducehuman immunoglobulin production substantially in the SCID-HuPBMCtransfer model. As shown in FIG. 19A, Luc90.H1 held down the increase inIgG production in PBS and isotype control as early as Day 4 (4 daysafter the treatment with the first dose of the antibody). This reductioncontinued throughout the 7 weeks (Day 32) of the test period. Forexample, at Day 18, the human IgG production increased by 225% in IgG2bisotype control, by 181% in the PBS control, while human IgG productiondecreased by 14% with Luc90H.1 treatment. Luc90H.1 not only abolishedthe 181-225% increase in the human IgG production in the control groups,but also resulted in an additional 14% decrease in the IgG production.At Day 25, Luc90H.1 not only abolished the 3 fold increase in human IgGproduction in the control groups but also gave an additional 24%decrease in human IgG production.

Luc 63.2 also effectively reduced IgG production in vivo. As shown inFIG. 19B, Luc63.2 abolished the 37-46% increase in human IgG productionin the control groups (PBS and IgG2a isotype control) and gave rise toan additional 59% decrease in IgG production. In this same study,Luc90.H1 was compared with Luc63.2 and Luc90.H1 abolished the 37-114%increase in the control groups (PBS and IgG2b isotype control) and gavean additional 14% decrease in IgG production by mice engrafted withhuman peripheral blood mononuclear cells (PBMCs).

FIG. 19C further summarizes the reduction in the Ig production by Luc90and Luc63 treatment in the SCIDHuPBMC model. While abolishing theincrease of IgG production in mice treated with isotype and PBScontrols, Luc90 caused an additional decrease in IgG production by 14%,22%, 24%,and 39%, and Luc63 had additional decrease by 40% and 59%.Thus, we can conclude that anti-Luc treatment of SCID mice engraftedwith human PBMCs (SCID-HuPBMC) not only completely abolishes theincrease in human immunoglobulin normally observed in the serum of theseanimals, but also gives an additional decrease compared to pretreatmentlevels.

Example 11 ADCC Activities of Anti-CS1 Antibodies Effector CellsPreparation:

Human peripheral blood mononuclear cells (PBMCs) (effector cells) wereisolated from whole blood using standard density Ficoll-Paque (AmershamBiosciences) gradients. Cells were washed and resuspended in RPMI mediumsupplemented with 1% bovine serum albumin (BSA).

Target Cells Preparation:

Stable transfectant cells expressing cell surface CS-1 (target cells)were washed and resuspended in RPMI medium supplemented with 1% BSA.Cells were plated at 100,000 cells/well in 50 μl total volume. Mouseanti-human CS-1 monoclonal antibodies (Luc90.H1 or Luc63.2.22) orisotype control antibodies (mouse IgG2b or IgG2a, respectively) wereadded at various concentrations to the target cells in a final volume of100 μl, and incubated for 30 minutes at room temperature.

After incubation, 100 μl of effector PBMCs were added to the targetcells at a 20:1 ratio in 200 μl final volume. Target and effector cellswere incubated at 37° C. for 5 hours or overnight. Cells werecentrifuged at 350×g for 5 minutes, and 100 μl/well of supernatant wascollected and transferred into an optically clear 96-well flat bottommicrotiter plate.

Lactate Dehydrogenase Assay:

To determine the lactate dehydrogenase (LDH) activity contained in thesupernatants, 100 μl reaction mixture from Cytotoxicity Detection Kit(Roche Applied Science, Indianapolis, Ind.) was added to each well, andsamples were incubated for up to 30 minutes at 15-25° C. During thisincubation period, the microtiter plate was protected from light. Theabsorbance of the samples was measured at 490 nm using an ELISA reader.

To determine the percentage of cell-mediated cytotoxicity, the averageabsorbance of the samples was calculated and background controls weresubtracted using the following equation:

$\mspace{20mu} {{{Cytotoxicity}\mspace{14mu} (\%)} = {\frac{{{LDH}\mspace{14mu} {release}_{sample}} - {SR}_{effector} - {SR}_{target}}{{MR}_{target} - {SR}_{target}}100}}$SR:  Spontaneous  Release MR:  Maximum  Release

The experimental controls were spontaneous release of the target cellsalone or the effector cells alone. The target cells were assayed in 2%Triton-X 100 (1:1) solution.

Anti-CS1 Antibodies Induce Antibody-Derived Cytotoxicity (ADCC)

The experiment showed that anti-CS1 antibodies Luc63.2 and Luc90 inducedantibody-derived cytotoxicity (ADCC) of cells expressing CS1 in thepresence of PBMCs (the effector cells). As shown in FIG. 20, Luc90induces cytotoxicity in a dosage-dependent manner. An amount of 50 μg/mlof Luc90 induced almost 50% cytotoxicity of the target cells. Luc63.2generally induced 60-80% cytotoxicity of the target cells with a doserange of 10-50 μg/ml. Similar results were obtained from experimentsconducted with two additional donors.

Example 12 ADCC Activity with Low Fucose CS1 Antibodies

Cloning of Luc90 Variable Region cDNAs

The murine variable regions (sequence ID #3 and #4) were cloned from theLuc90 hybridoma cell line by standard methods. Briefly, total RNA wasextracted and double-stranded cDNA was synthesized using the SMART5′-RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto,Calif.) following the supplier's protocol. PCR fragments of the variableregion cDNAs were cloned into the pCR4Blunt-TOPO vector (InvitrogenCorporation, Carlsbad, Calif.) for sequence determination. Severalplasmid clones were sequenced for each of the heavy and light chains.Unique sequences homologous to typical mouse heavy and light chainvariable regions were identified.

Construction of Chimeric Luc90 VH and VL Expression Vectors

A gene encoding each of Luc90 VH and VL was designed as a mini-exonincluding a signal peptide, a splice donor signal, Kozak initiationsequence and appropriate restriction enzyme sites for subsequent cloninginto a mammalian expression vector. Primers were designed to contain theappropriate restrictions sites and complementarity for PCR from the TOPOvectors containing either the VH or VL genes. The PCR-amplifiedfragments were purified by Qiaquick PCR purification kit (Qiagen) anddigested with MluI and XbaI. The Luc90 VH gene was subcloned intopHuHCg1.D (wildtype) or pHuHCg1.D.AA (BS mutant) to create the plasmidspChiHuHCg1.D-MuLuc90VH and pChiHuHCg1.D.AA-MuLuc90VH, respectively. TheBS mutant contains two amino acid changes (L234A/L235A) in the CH2region of IgG1, such that binding to Fc receptors is abolished (Xu etal., (2000) Cell Immunol. 200:16-26). The Luc90 VL gene was subclonedinto pVk to create the plasmid pChiVk-MuLuc90VL. Single plasmidexpression vectors were created such that the heavy and light chaingenes could be expressed from a single plasmid. The heavy chain vectorswere digested with EcoRI to remove the entire heavy chain region andsubcloned into a single EcoRI sight in the light chain vector. The BSmutant heavy chain was combined with pChiVk-MuLuc90VL vector fragment tocreate the plasmid pChiLuc90-BSK while the wildtype heavy chain wassubcloned into pChiVk-MuLuc90VL vector to create the plasmidpChiLuc90-g1K.

Expression of Chimeric Luc90

Chimeric Luc90 IgG1/κ wildtype and BS antibodies were produced by stabletransfection of Sp2/0 cells with the pChiLuc90-g1K and pChiLuc90-BSKvectors, respectively. A low-fucose antibody was produced by stabletransfection of YB2/0 cells with the pChiLuc90-g1K vector. Positiveclones were selected for with mycophenolic acid media and screened byELISA. The wildtype clone AH4, BS mutant HG12 and low-fucose clone 5E4were selected for high expression, adapted to Gibco Hybridoma serum freemedia with 2% low Ig Fetal Bovine Serum. Two liter cultures were grownin roller bottles for purification. Antibodies were purified by standardProtein-G affinity column chromatography.

FIGS. 21A-C depict data on effect of low fucose antibodies incytotoxicity assays CS1 expressing cells (stable transfectant and humanmultiple myeloma cell lines) were treated with anti-CS1 Luc90 chimericantibodies (both wild type and antibodies modified with decreased levelsof fucose). Anti-CS1 Luc90 chimeric antibodies stimulateantibody-dependent cellular cytotoxicity of cells expressing CS1. (FIG.21A shows cytotoxicity of a stable cell line expressing human CS1; FIGS.21B and 21C depict cytotoxicity of two human myeloma cell lines, OPM2(FIG. 21B) and L363 (FIG. 21C). In each case, cytotoxicity issignificantly enhanced by antibodies which have low levels of fucose(through growth in YB2/0 cells as detailed above.)

Example 13 Treatment of Myeloma with Anti-CS1 Antibodies

Treatment with anti-CS1 antibody in vivo was performed on a myelomamouse tumor model by injecting antibody intraperitoneally into the testsubject. As shown in FIG. 22, anti-CS1 antibody treatment (Luc63 andLuc90) decreases tumor size compared to isotype control treated animals.In this study, 1×10⁷ myeloma cells (L363 myeloma cell line) were i.p.injected into CB.17 SCID mice. Two weeks later, when tumor size reached˜80 mm3, mice were randomized into 4 groups with 8 mice per group. Micewere treated with anti-CS1 antibodies (Luc63 or Luc90) or isotypecontrol antibodies (mouse IgG2a or mouse IgG2b). Mice were dosed with200 μg antibody/mouse for 8 doses at 3 doses per week. The results showthat mice treated with anti-CS1 antibodies have significantly reducedtumor volumes compared to isotype control antibody treated mice. By day25 of the study (after 5 doses), Luc 63 treated mice show average tumorsize ˜100 mm³ compared to IgG2a isotype control antibody treated mice(average tumor size ˜800 mm³.) Luc 90 treated mice show average tumorsize ˜400 mm³ compared to IgG2b isotype control antibody treated mice(which have average tumor size ˜950 mm³.) Mice treated with anti-CS1Luc63 have no measureable tumors for up to 2.5 weeks post treatment,pointing to the striking efficacy of the antibody at eliminatingtumorigenic cells.

Additional model systems for myeloma include SCID mice implantedintravenously (i.v.), intraperitoneally (i.p.) or directly injected intothe bone (orthotopically) with fluorescently-labeled or unlabeledmyeloma or mature B-cell lines, e.g. ARH77, CESS, IM9, L363, LP1 andOPM2. These lines will be used to test the effects of antagonisttreatment in myeloma animal model systems. These cell lines express theantigen recognized by anti-human CS1 antibodies. Animals are randomizedinto groups and subjected to a treatment regimen with anti-human CS1antibodies or control antibodies (for example, isotype controlantibodies). Antibodies are administered at several dosage levels, forexample a dose of 1-10 mg/kg for a total of 9-10 doses givenintraperitoneally every 3-4 days. Tumor size is measured twice weeklyfor 35-40 days for each treatment group. Clinical manifestations ofmyeloma are noted. Dates of death are recorded for each mouse.

Animal studies will also be initiated to determine the potential synergybetween anti-CS1 antibody treatment and chemotherapy. Xenograft tumorsare allowed to grow until they reach an approximate size of between50-100 mm³, and for mice injected i.v., i.p. or orthotopically, cancercells are allowed to engraft in animals. At that time, animals arerandomized into groups and subjected to a treatment regimen withanti-human CS1 antibodies or control antibodies (for example, isotypecontrol antibodies). Alternatively, animals may be subjected totreatment with anti-human CS1 antibodies or control antibodies (forexample, isotype control antibodies) in combination with standardchemotherapy agents, including combinations of prednisone and melphalanor other alkylating agents (e.g. cyclophosphamide or chlorambucil), orvincristine, doxorubicin and high-dose dexamethasone (VAD) treatment, orother chemotherapy regimens known to those of skill in the art.Antibodies are administered at several dosage levels, for example a doseof 1-10 mg/kg for a total of 9-10 doses given intraperitoneally every3-4 days. Chemotherapy is administered intraperitoneally every 3-4 daysat an effective concentration, for example 1 mg/kg or other effectivedose that is known to those of skill in the art. Tumor size (fors.c.injected animals) is measured twice weekly for 35-40 days for eachtreatment group. Clinical manifestations of myeloma are noted, includingserum immunoglobulin in mice injected with cell lines that secrete humanimmunoglobulin (IM9, CESS, ARH-77, and LP-1). Dates of death arerecorded for each mouse. The efficacy of antibody treatment in thepresence and absence of chemotherapy will be evaluated.

It is understood that the examples described above in no way serve tolimit the true scope of this invention, but rather are presented forillustrative purposes. All publications, sequences of accession numbers,and patent applications cited in this specification are hereinincorporated by reference as if each individual publication, accessionnumber, or patent application were specifically and individuallyindicated to be incorporated by reference.

DEPOSIT OF HYBRIDOMA: The hybridoma cell line Luc90, secretingmonoclonal antibody Luc90, was deposited with the American Type CultureCollection (“ATCC”), at P.O. Box. 1549, Manassas, Va. 20108, on Mar. 26,2003, in compliance with the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure (“Budapest Treaty”) on behalf of the Assignee, Protein DesignLabs, Inc. (now PDL Biopharma, Inc.). The deposited hybridoma wasassigned ATCC accession number PTA-5091. The hybridoma cell lineLuc63.2.22, secreting monoclonal antibody Luc63, was deposited with theATCC at P.O. Box. 1549, Manassas, Va. 20108, on May 6, 2004, incompliance with the Budapest Treaty on behalf of the Assignee, ProteinDesign Labs, Inc. (which became PDL Biopharma, Inc.). The depositedhybridoma was assigned ATCC accession number PTA-5950.

TABLE 2 SEQ ID NO: 1 PDL primekey:433671     DNA Sequence Nucleic AcidAccession #:   NM_021181 GI:19923571|ref|NM_021181.3| Homo sapiens SLAMfamily member 7 (SLAMF7), mRNA 1 cttccagaga gcaatatggc tggttccccaacatgcctca ccctcatcta tatcctttgg 61 cagctcacag ggtcagcagc ctctggacccgtgaaagagc tggtcggttc cgttggtggg 121 gccgtgactt tccccctgaa gtccaaagtaaagcaagttg actctattgt ctggaccttc 181 aacacaaccc ctcttgtcac catacagccagaagggggca ctatcatagt gacccaaaat 241 cgtaataggg agagagtaga cttcccagatggaggctact ccctgaagct cagcaaactg 301 aagaagaatg actcagggat ctactatgtggggatataca gctcatcact ccagcagccc 361 tccacccagg agtacgtgct gcatgtctacgagcacctgt caaagcctaa agtcaccatg 421 ggtctgcaga gcaataagaa tggcacctgtgtgaccaatc tgacatgctg catggaacat 481 ggggaagagg atgtgattta tacctggaaggccctggggc aagcagccaa tgagtcccat 541 aatgggtcca tcctccccat ctcctggagatggggagaaa gtgatatgac cttcatctgc 601 gttgccagga accctgtcag cagaaacttctcaagcccca tccttgccag gaagctctgt 661 gaaggtgctg ctgatgaccc agattcctccatggtcctcc tgtgtctcct gttggtgccc 721 ctcctgctca gtctctttgt actggggctatttctttggt ttctgaagag agagagacaa 781 gaagagtaca ttgaagagaa gaagagagtggacatttgtc gggaaactcc taacatatgc 841 ccccattctg gagagaacac agagtacgacacaatccctc acactaatag aacaatccta 901 aaggaagatc cagcaaatac ggtttactccactgtggaaa taccgaaaaa gatggaaaat 961 ccccactcac tgctcacgat gccagacacaccaaggctat ttgcctatga gaatgttatc 1021 tagacagcag tgcactcccc taagtctctgctcaaaaaaa aaacaattct cggcccaaag 1081 aaaacaatca gaagaattca ctgatttgactagaaacatc aaggaagaat gaagaacgtt 1141 gacttttttc caggataaat tatctctgatgcttctttag atttaagagt tcataattcc 1201 atccactgct gagaaatctc ctcaaacccagaaggtttaa tcacttcatc ccaaaaatgg 1261 gattgtgaat gtcagcaaac cataaaaaaagtgcttagaa gtattcctat agaaatgtaa 1321 atgcaaggtc acacatatta atgacagcctgttgtattaa tgatggctcc aggtcagtgt 1381 ctggagtttc attccatccc agggcttggatgtaaggatt ataccaagag tcttgctacc 1441 aggagggcaa gaagaccaaa acagacagacaagtccagca gaagcagatg cacctgacaa 1501 aaatggatgt attaattggc tctataaactatgtgcccag cactatgctg agcttacact 1561 aattggtcag acgtgctgtc tgccctcatgaaattggctc caaatgaatg aactactttc 1621 atgagcagtt gtagcaggcc tgaccacagattcccagagg gccaggtgtg gatccacagg 1681 acttgaaggt caaagttcac aaagatgaagaatcagggta gctgaccatg tttggcagat 1741 actataatgg agacacagaa gtgtgcatggcccaaggaca aggacctcca gccaggcttc 1801 atttatgcac ttgtgctgca aaagaaaagtctaggtttta aggctgtgcc agaacccatc 1861 ccaataaaga gaccgagtct gaagtcacattgtaaatcta gtgtaggaga cttggagtca 1921 ggcagtgaga ctggtggggc acggggggcagtgggtactt gtaaaccttt aaagatggtt 1981 aattcattca atagatattt attaagaacctatgcggccc ggcatggtgg ctcacacctg 2041 taatcccagc actttgggag gccaaggtgggtgggtcatc tgaggtcagg agttcaagac 2101 cagcctggcc aacatggtga aaccccatctctactaaaga tacaaaaatt tgctgagcgt 2161 ggtggtgtgc acctgtaatc ccagctactcgagaggccaa ggcatgagaa tcgcttgaac 2221 ctgggaggtg gaggttgcag tgagctgagatggcaccact gcactccggc ctaggcaacg 2281 agagcaaaac tccaatacaa acaaacaaacaaacacctgt gctaggtcag tctggcacgt 2341 aagatgaaca tccctaccaa cacagagctcaccatctctt atacttaagt gaaaaacatg 2401 gggaagggga aaggggaatg gctgcttttgatatgttccc tgacacatat cttgaatgga 2461 gacctcccta ccaagtgatg aaagtgttgaaaaacttaat aacaaatgct tgttgggcaa 2521 gaatgggatt gaggattatc ttctctcagaaaggcattgt gaaggaattg agccagatct 2581 ctctccctac tgcaaaaccc tattgtagtaaaaaagtctt ctttactatc ttaataaaac 2641 agatattgtg agattcaaaa aaaaaaaaaaaa SEQ ID NO: 2 Amino Acid Sequence - CS1GI:19923571|ref|NM_021181.3| Homo sapiens SLAM family member 7 (SLAMF7)MAGSPTCLTLIYILWQLTGSAASGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSMVLLCLLLVPLLLSLFVLGLFLWFLKRERQEEYIEEKKRVDICRETPNICPHSGENTEYDTIPHTNRTILKEDPANTVYSTVEIPKKMENPHSLLTMPDTPRLFAYENVI

TABLE 4 Amino Acid Sequences of CS1 Antibodies Luc-90 VH - SEQ ID NO: 3QVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEW                SEQ IDNO: 9  SEQ ID NO: 10 IGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSS     SEQ ID NO: 11 Luc-90 VL - SEQ ID NO: 4DIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLL             SEQ ID NO:12  SEQ ID NO: 13 IYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYST                              SEQ ID NO: 14 PLTFGAGTKLELK Luc-63 VH -SEQ ID NO: 5 EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEW               SEQ ID NO: 15 SEQ ID NO: 16IGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSS    SEQ ID NO: 17 Luc-63 VL - SEQ ID NO: 6DIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLL                       SEQ ID NO: 18IYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFC  SEQ ID NO: 19QQYSSYPYTFGGGTKLEIK SEQ ID NO: 20 Luc-34 VH - SEQ ID NO: 7QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEW                 SEQ IDNO: 21 SEQ ID NO: 22 IGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSA     SEQ ID NO: 23 Luc-34 VL - SEQ ID NO:8DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLL              SEQ ID NO:24 SEQ ID NO: 25 ISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK SEQ ID NO: 26

TABLE 5 Anti-CS1 Luc63 Variable Heavy Chain Region PutativeGlycosylation Site Luc-63 VH (SEQ ID NO:27)

Luc-63 VL (SEQ ID NO:28)

TABLE 6 Luc63 (NYA) Humanization - Alignment of the VH regions ofMuLuc63 (SEQ ID NO: 37), human variable region cDNA (SEQ ID NO: 30),human JH1 cDNA (SEQ ID NO: 40), and huLuc63 (SEQ ID NO: 41), and VLregions of MuLuc63 (SEQ ID NO: 42), human variable region cDNA (SEQ IDNO: 43) and huLuc63 (SEQ ID NO: 44). MuLuc-63 VHEVKLLESGGGLVQPGGSLKLSCAASGFDFS RYWMS HumanVH cDNAEVQLVESGGGLVQPGGSLRLSCAASGFTFS HuLuc-63 VHEVQLVESGGGLVQPGGSLRLSCAASGFDFS RYWMS MuLuc-63 VH WVRQAPGKGLEWIGEINPDSSTINYTPSLKD HumanVH cDNA WVRQAPGKGLEWVA HuLuc-63 VHWVRQAPGKGLEWIG EINPDSSTINYAPSLKD MuLuc-63 VHKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR Human JH1 cDNARFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR HuLuc-63 VHKFIISRDNAKNSLYLQMNSLRAEDTAVYYCAR MuLuc-63 VH PDGNYWYFDV WGAGTTVTVSSHuman JH1 cDNA HuLuc-63 VH PDGNYWYFDV WGQGTLVTVSS MuLuc-63 VLDIVMTQSHKFMSTSVGDRVSITC KASQDVGIAVA HumanVL cDNA DIQMTQSPSSLSASVGDRVTITCHuLuc-63 VL DIQMTQSPSSLSASVGDRVTITC KASQDVGIAVA MuLuc-63 VLWYQQKPGQSPKLLIY WASTRHT HumanVL cDNA WYQQKPGKVPKLLIY HuLuc-63 VLWYQQKPGKVPKLLIY WASTRHT MuLuc-63 VL GVPDRFTGSGSGTDFTLTISNVQSEDLADYFCHumanVL cDNA GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC HuLuc-63 VLGVPDRFSGSGSGTDFTLTISSLQPEDVATYYC MuLuc-63 VL QQYSSYPYT FGGGTKLEIKHumanVL cDNA           FGQGTKVEIK HuLuc-63 VL QQYSSYPYT FGQGTKVEIK

TABLE 7 Alignment of the VH regions of MuLuc63 (SEQ ID NO: 45), E55 3-14(SEQ ID NO: 46), HuLuc63 (SEQ ID NO: 47)         1         2         3         4         0         0         0         0 MuLucEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPG 63 E55 3-EVQLVESGGGLVQPGGSLRLSCAASGFTFS-----WVRQAPG 14 HuLucEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPG 63       5      6             7         8       0  a   0             0         0  a MuLuc-KGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMS 63 E55KGLEWVA-----------------RFTISRDNAKNSLYLQMN 3-14 HuLuc-KGLEWIGEINPDSSTINY A PSLKDKFIISRDNAKNSLYLQMN 63                   1           1          9         0           1bc       0         0ab         0 MuLuc-KVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSS 63 E55 3-SLRAEDTAVYYCAR----------WGQGTLVTVSS 14/JH1 HuLuc-SLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS 63

TABLE 8 Alignment of the VL region of MuLuc63 (SEQ ID NO: 48), III-2R(SEQ ID NO: 49) and HuLuc63 (SEQ ID NO: 50) antibody amino acidsequences          1         2         3         4         0         0         0         0 MuLucDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQ 63 III-2RDIQMTQSPSSLSASVGDRVTITC-----------WYQQKPGK HuLucDIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGK 63       5         6         7         8       0         0         0         0 MULUCSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLA 63 III-2RVPKLLIY-------GVPSRFSGSGSGTDFTLTISSLQPEDVA HuLuc VPKLLIYWASTRHTGVP DRFSGSGSGTDFTLTISSLQPEDVA 63    1      9         0      0         0 MULUCDYFCQQYSSYPYTFGGGTKLEIK 63 III-2R TYYC---------FGQGTKVEIK HuLucTYYCQQYSSYPYTFGQGTKVEIK 63

TABLE 9 Oligonucleotides Used for the Synthesis of the HuLuc63 VH and VLgene HuLuc63 VH Gene Oligonucleotide 1 (SEQ ID NO: 53)5′-TTTACGCGTCCACCATGGATTTTGGGCTGATTT-3′ Oligonucleotide 2 (SEQ ID NO:54) 5′-TTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGT-3′ Oligonucleotide 3(SEQ ID NO: 55) 5′-GCAGCTTGTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGA-3′Oligonucleotide 4 (SEQ ID NO: 56)5′-GGATCCCTGAGACTCTCCTGTGCAGCCTCAGGATTCGATT-3′ Oligonucleotide 5 (SEQ IDNO: 57) 5′-TTAGTAGATATTGGATGAGTTGGGTCCGGCAGGCTCCAGG-3′ Oligonucleotide 6(SEQ ID NO: 58) 5′-GAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGC-3′Oligonucleotide 7 (SEQ ID NO: 59)5′-AGTACGATAAACTATGCTCCATCTCTAAAGGATAAATTCA-3′ Oligonucleotide 8 (SEQ IDNO: 60) 5′-TCATCTCCAGAGACAACGCCAAAAATAGCCTGTACCTGCA-3′ Oligonucleotide 9(SEQ ID NO: 61) 5′-AATGAACAGCCTCAGAGCTGAGGACACAGCCGTTTATTAC-3′Oligonucleotide 10 (SEQ ID NO: 62)5′-TGTGCAAGACCGGACGGAAACTACTGGTACTTCGATGTCT-3′ Oligonucleotide 11 (SEQID NO: 63) 5′-GGGGCCAGGGGACCCTCGTCACCGTCTCCTCAGGTAAGAA-3′Oligonucleotide 12 (SEQ ID NO: 64)5′-TTTTCTAGAGGCCATTCTTACCTGAGGAGACGGT-3′ Oligonucleotide 13 (SEQ ID NO:65) 5′-GACGAGGGTCCCCTGGCCCCAGACATCGAAGTACCAGTAG-3′ Oligonucleotide 14(SEQ ID NO: 66) 5′-TTTCCGTCCGGTCTTGCACAGTAATAAACGGCTGTGTCCT-3′Oligonucleotide 15 (SEQ ID NO: 67)5′-CAGCTCTGAGGCTGTTCATTTGCAGGTACAGGCTATTTTT-3′ Oligonucleotide 16 (SEQID NO: 68) 5′-GGCGTTGTCTCTGGAGATGATGAATTTATCCTTTAGAGAT-3′Oligonucleotide 17 (SEQ ID NO: 69)5′-GGAGCATAGTTTATCGTACTGCTATCTGGATTAATTTCTC-3′ Oligonucleotide 18 (SEQID NO: 70) 5′-CAATCCATTCTAGCCCTTTCCCTGGAGCCTGCCGGACCCA-3′Oligonucleotide 19 (SEQ ID NO: 71)5′-ACTCATCCAATATCTACTAAAATCGAATCCTGAGGCTGCA-3′ Oligonucleotide 20 (SEQID NO: 72) 5′-CAGGAGAGTCTCAGGGATCCTCCAGGCTGCACCAGGCCAC-3′Oligonucleotide 21 (SEQ ID NO: 73)5′-CTCCAGACTCGACAAGCTGCACCTCACACTGGACCCCTTT-3′ Oligonucleotide 22 (SEQID NO: 74) 5′-TAAAAGAGCAACAATAAAAAAAATCAGCCCAAAATCCATG-3′ HuLuc63 VLGene Oligonucleotide A (SEQ ID NO: 75)5′-TTTACGCGTCCACCATGGAGACACATTCTCAGGTCTTTGTAT A-3′ Oligonucleotide B(SEQ ID NO: 76) 5′-CATGTTGCTGTGGTTGTCTGGTGTTGAAGGAGACATTCAG-3′Oligonucleotide C (SEQ ID NO: 77)5′-ATGACCCAGTCTCCTTCATCACTTTCCGCATCAGTAGGAG-3′ Oligonucleotide D (SEQ IDNO: 78) 5′-ACAGAGTCACTATCACCTGCAAGGCCAGTCAGGATGTGGG-3′ Oligonucleotide E(SEQ ID NO: 79) 5′-TATTGCTGTAGCCTGGTATCAACAGAAACCAGGGAAAGTA-3Oligonucleotide F (SEQ ID NO: 80)5′-CCTAAACTATTGATTTACTGGGCATCCACCCGGCACACTG-3′ Oligonucleotide G (SEQ IDNO: 81) 5′-GAGTCCCTGATCGATTCTCAGGCAGTGGATCTGGGACAGA-3′ Oligonucleotide H(SEQ ID NO: 82) 5′-TTTCACTCTCACCATTAGCTCACTACAGCCTGAAGACGTG-3′Oligonucleotide I (SEQ ID NO: 83)5′-GCAACTTATTACTGTCAGCAATATAGCAGCTATCCATACA-3′ Oligonucleotide J (SEQ IDNO: 84) 5′-CGTTCGGACAGGGGACCAAGGTGGAAATCAAACGTAAGTG-3′ Oligonucleotide K(SEQ ID NO: 85) 5′-TTTTCTAGATTAGGAAAGTGCACTTACGTTTGATTTCCAC-3′Oligonucleotide L (SEQ ID NO: 86)5′-CTTGGTCCCCTGTCCGAACGTGTATGGATAGCTGCTATAT-3′ Oligonucleotide M (SEQ IDNO: 87) 5′-TGCTGACAGTAATAAGTTGCCACGTCTTCAGGCTGTAGTG-3′ Oligonucleotide N(SEQ ID NO: 88) 5′-AGCTAATGGTGAGAGTGAAATCTGTCCCAGATCCACTGCC-3′Oligonucleotide O (SEQ ID NO: 89)5′-TGAGAATCGATCAGGGACTCCAGTGTGCCGGGTGGATGCC-3′ Oligonucleotide P (SEQ IDNO: 90) 5′-CAGTAAATCAATAGTTTAGGTACTTTCCCTGGTTTCTGTT-3′ Oligonucleotide Q(SEQ ID NO: 91) 5′-GATACCAGGCTACAGCAATACCCACATCCTGACTGGCCTT-3′Oligonucleotide R (SEQ ID NO: 92)5′-GCAGGTGATAGTGACTCTGTCTCCTACTGATGCGGAAAGT-3′ Oligonucleotide S (SEQ IDNO: 93) 5′-GATGAAGGAGACTGGGTCATCTGAATGTCTCCTTCAACAC-3′ Oligonucleotide T(SEQ ID NO: 94) 5′-CAGACAACCACAGCAACATGTATACAAAGACCTGAGAATG-3′

1. An isolated antibody or antigen binding fragment thereof which bindsto a CS1 protein encoded by SEQ ID NO:1, wherein said antibody isproduced by a hybridoma cell line having ATCC designation numberPTA-5091.
 2. An isolated antibody or antigen binding fragment thereofwhich binds to a CS1 protein encoded by SEQ ID NO:1, wherein saidantibody is produced by a hybridoma cell line deposited under ATCCpatent designation number PTA-5950.
 3. An antibody-drug conjugatecomprising the isolated antibody or antigen binding fragment of claim 1conjugated to a cytotoxic agent.
 4. An antibody-drug conjgate comprisingthe isolated antibody or antigen binding fragment of claim 2 conjugatedto a cytotoxic agent.